Uses of a dual v region antibody-like protein

ABSTRACT

Uses of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region.

BACKGROUND OF THE INVENTION

Interleukin-4 (IL-4) is a pleiotropic cytokine that has a broad spectrum of biological effects on lymphoid B and T cells, and many non-lymphoid cells including monocytes, endothelial cells and fibroblasts. For example, IL-4 stimulates the proliferation of several IL-2- and IL-3-dependent cell lines, induces the expression of class II major histocompatability complex molecules on resting B cells, and enhances the secretion of IgG4 and IgE by human B cells. IL-4 is associated with a Th2-type immune response, and is produced by and promotes differentiation of Th2 cells. IL-4 has been implicated in a number of disorders, such as allergy and asthma.

IL-13 is a recently identified (Minty, A. et al., Nature, 1993, 362, 248-250, and McKenzie, A. N. et al., Proc. Natl. Acad. Sci. U.S.A, 1993, 90, 3735-3739) cytokine of 112 amino acids secreted by the activated T lymphocytes, the B lymphocytes and the mastocytes after activation. By virtue of its numerous biological properties shared with IL-4, IL-13 has been described as an IL-4-like cytokine. Its activities are indeed similar to those of IL-4 on the B cells (Defrance, T. et al., J. Exp. Med., 1994, 179, 135-143, Punnonen, J. et al., Proc. Natl. Acad. Sci. (USA), 1993, 90, 3730-3734, Fior, R. et al., Eur. Cytokine Network, 1994, 5, 593-600), the monocytes (Muzio, M. R. F. et al., Blood, 1994, 83, 1738-1743, De Waal Malefyt, R. et al., J. Immunol, 1993, 151, 6370-6381, Doyle, A. et al., Eur. J. Immunol. 1994, 24, 1441-1445, Montaner, L. J. et al., J. Exp. Med., 1993, 178, 743-747, Sozzani, P. et al., J. Biol. Chem., 1995, 270, 5084-5088) and other non-haematopoietic cells (Herbert, J. M. et al., Febs Lett., 1993, 328, 268-270, and Derocq, J. M. et al., Febs Lett. 1994, 343, 32-36). On the other hand, contrary to IL-4, it does not exert a specific effect on resting or activated T cells (Zurawuki, G. et al., Immunol. Today, 1994, 15, 19-26).

Various biological activities of IL-13 on the monocytes/macrophages, the B lymphocytes and certain haematopoietic precursors have been described in detail by A. J. Minty as well as in review articles on IL-13. Several data indicate, in addition, that this cytokine has a pleiotropic effect on other cell types. These non-haematopoietic cells which are directly affected by IL-13 are endothelial and microglial cells, keratinocytes and kidney and colon carcinomas.

One of the stages in the analysis of the signal transmitted by a biological molecule within a cell consists in identifying its membrane receptor. The research studies carried out to this end on the IL-13 receptor have shown that IL-13 and IL-4 have a common receptor, or at the very least some of the components of a common receptor complex, as well as common signal transduction elements (Zurawski S. M. et al., Embo Journal, 1993, 12, 2663-2670, Aversa, G. et al., J. Exp. Med., 1993, 178, 2213-2218, Vita, N. et al., Biol. Chem., 1995, 270, 3512-3517, Lefort, S. et al., Febs Lett., 1995, 366, 122-126). This receptor is present at the surface of various cell types, in a variable number according to the cell type considered. The comparative distribution of the IL-13 and IL-4 receptors has been indicated by A. J. Minty (Interleukin-13 for Cytokines in Health and Disease. Eds D. G. Remick and J. S. Frie, Marcel Decker, N.Y. 1996).

The cell surface receptors and receptor complexes bind IL-4 and/or IL-13 with different affinities. The principle components of receptors and receptor complexes that bind IL-4 and/or IL-13 are IL-4Rα, IL-13RαI and IL-13Rα2. These chains are expressed on the surface of cells as monomers or heterodimers of IL-4Rα/IL-13RαI (Type II IL-4R) or IL-4Rα/c (Type I IL-4R). IL-4Ra monomer and IL-4R/c heterodimer bind IL-4, but not IL-13. IL-13Rα1 and IL-13Rα2 monomers bind IL-13, but do not bind IL-4. IL-4Rα/IL-13Rα1 heterodimer binds both IL-4 and IL-13 (Murata et al., Int. J. Hematol., 1999, 69, 13-20).

Th2-type immune responses promote antibody production and humoral immunity, and are elaborated to fight off extracellular pathogens. Th2 cells are mediators of Ig production (humoral immunity) and produce IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13 (Tanaka, et, al., Cytokine Regulation of Humoral Immunity, 251-272, Snapper, ed., John Wiley and Sons, New York (1996)). Th2-type immune responses are characterized by the generation of certain cytokines (e.g., IL-4, IL-13) and specific types of antibodies (IgE, IgG4) and are typical of allergic reactions, which may result in watery eyes and asthmatic symptoms, such as airway inflammation and contraction of airway muscle cells in the lungs.

Both IL-4 and IL-13 are therapeutically important cytokines based on their biological functions and play critical roles in many diseases, including asthma (Curr Opin Allergy Clin Immunol 2005, Vo. 5, 161-166). IL-4 has been shown to be able to inhibit autoimmune disease and IL-4 and IL-13 have both shown the potential to enhance anti-tumor immune responses. Elevations in IL-4 and IL-13 and their receptors have been linked to the pathogenesis of idiopathic pulmonary fibrosis (IPF) (Jakubzick C. et al., Am J Pathol. 2004:164(6):1989-2001; Murray L A et al. Int J Biochem Cell Biol. 2008:40(10):2174-82. Evidence in the literature demonstrate that the TH2 cytokines IL-4 and IL-13 play multiple roles in the pathogenesis of IPF as mediators of this lung tissue remodeling and fibrosis. Although the Th2-type CD4+t cells in the lung are likely the predominant sources of IL-4 and IL-13, and are implicated as important regulators of extracellular matrix remodeling (Wynn, T A, Nat. Rev. Immunol, 4:583-594, 2004), other cell types including mast cells, basophils, eosinophils, macrophages and epithelial cells may also be potential sources of these cytokines (Gordon S and Martinez F O, Immunity Rev. 32:593-604, 2010). In IPF patients, IL-13 and IL-4 levels in bronchial alveolar lavage fluid are elevated compared to normal controls. Such evidence suggests that therapies capable of suppressing or neutralizing these cytokines have the potential for delaying the progression of fibrosis in IPF patients. Since both cytokines are involved in the pathogenesis of allergic diseases or fibrotic diseases, inhibitors of these cytokines could provide therapeutic benefits.

Accordingly, a need exists for improved agents that inhibit IL-4, inhibit IL-13, and single agents that inhibit both IL-4 and IL-13 that are non-immunogenic and safe for use in humans. We previously reported on a dual V region antibody like binding peptide having four binding sites that specifically bind to IL-4 and IL-13 (WO2009/052081 (PCT/US2008/079787), which is incorporated by reference in its entirety.

SUMMARY OF THE INVENTION

An embodiment of the invention is a maximal safe therapeutic dose of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 to a human subject having an area under the plasma concentration versus time curve calculated using the trapezoidal method from time zero to real time (AUC_(last)) from about 433 ug·h/ml to about 14200 ug·h/ml. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the safe therapeutic dose is equal to or less than about 300 mg. In a further embodiment, the safe therapeutic dose is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

An embodiment of the invention is a maximal safe therapeutic dose of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 to a human subject having an area under the plasma concentration versus time curve extrapolated to infinity (AUC) from about 459 ug·h/ml to about 670014500 ug·h/ml. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the safe therapeutic dose is equal to or less than about 300 mg. In a further embodiment, the safe therapeutic dose is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

An embodiment of the invention is a maximal safe therapeutic dose of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 to a human subject having a maximum plasma concentration observed (C_(max)) from about 0.717 ug/ml to about 28.7 ug/ml. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the safe therapeutic dose is equal to or less than about 300 mg. In a further embodiment, the safe therapeutic dose is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

An embodiment of the invention is a maximal safe therapeutic dose of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 to a human subject having a first time to reach a maximum plasma concentration (t_(max)) from about 96 hr to about 168 hr. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the safe therapeutic dose is equal to or less than about 300 mg. In a further embodiment, the safe therapeutic dose is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

An embodiment of the invention is a maximal safe therapeutic dose of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 to a human subject having t_(last) from about 1679 hr to about 2020 hr. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the safe therapeutic dose is equal to or less than about 300 mg. In a further embodiment, the safe therapeutic dose is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

An embodiment of the invention is a maximal safe therapeutic dose of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 to a human subject having t_(1/2Z) from about 244 hr to about 536 hr. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the safe therapeutic dose is equal to or less than about 300 mg. In a further embodiment, the safe therapeutic dose is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

An embodiment of the invention is a maximal safe therapeutic dose of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 to a human subject having Vss/F from about 6830 ml to about 18770 ml. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the safe therapeutic dose is equal to or less than about 300 mg. In a further embodiment, the safe therapeutic dose is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

An embodiment of the invention is a maximal safe therapeutic dose of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 to a human subject having CL/F from about 12.1 ml/hr to about 38.4 ml/hr. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the safe therapeutic dose is equal to or less than about 300 mg. In a further embodiment, the safe therapeutic dose is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

An embodiment of the invention is a method of identifying or monitoring the occurrence of a safe therapeutic dose of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 having been administered to a human subject, said method comprising (a) administering a dose of said dual V region antibody-like protein or a fragment of a dual V region antibody-like region to said human subject; (b) measuring one or more events selected from the group consisting of intensive treatment in an emergency room or at home for allergic bronchospasm, blood dyscrasias, convulsions, alanine aminotransferase (ALT) >3×upper limit of normal range (ULN) associated with total bilirubin >2×ULN, asymptomatic ALT increase >10×ULN, development of drug dependency or drug abuse, ALT increase ≥2×ULN, hsCRP >10 mg/L for ≥72 hours, cardiac troponin I (cTnI) >2×ULN, a ventricular depolarization and repolarization time (QT) on an electrocardiogram (ECG) machine wherein the QT is automatically corrected by the ECG machine (QTc) that is QTc≥500 ms and severe skin reactions local to the site of IP injection; and (c) determining one or more said events as measured in (b) has not occurred wherein said dose is identified as said safe therapeutic dose having been administered to said human subject. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the safe therapeutic dose is equal to or less than about 300 mg. In a further embodiment, the safe therapeutic dose is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

An embodiment of the invention is a method monitoring whether a therapeutic dose of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 administered to a human subject is safe, said method comprising (a) administering said therapeutic dose of said dual V region antibody-like protein or a fragment of a dual V region antibody-like region to said human subject; (b) measuring one or more events selected from the group consisting of intensive treatment in an emergency room or at home for allergic bronchospasm, blood dyscrasias, convulsions, alanine aminotransferase (ALT) >3×upper limit of normal range (ULN) associated with total bilirubin >2×ULN, asymptomatic ALT increase >10×ULN, development of drug dependency or drug abuse, ALT increase ≥2×ULN, hsCRP >10 mg/L for ≥72 hours, cardiac troponin I (cTnI) >2×ULN, a ventricular depolarization and repolariztion time (QT) on an electrocardiogram (ECG) machine wherein the QT is automatically corrected by the ECG machine (QTc) that is QTc ≥500 ms and severe skin reactions local to the site of IP injection; and (c) determining one or more said events as measured in (b) has occurred wherein said therapeutic dose is identified as not safe and the therapeutic dose is discontinued or lowered. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the therapeutic dose is equal to or less than about 300 mg. In a further embodiment, the therapeutic dose is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

An embodiment of the invention is a method of selecting a safe therapeutic dose or of monitoring the safe use of a therapeutic dose of dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 to a human subject, said method comprising (a) administering a dose of said dual V region antibody-like protein or a fragment of a dual V region antibody-like region to said human subject; (b) measuring a level of C-reactive protein (CRP) in a blood sample from said human subject; and (c) determining said level of C-reactive protein (CRP) is less than 20 mg/L as measured in (b) wherein said dose is selected as said safe therapeutic dose to be administered to said human subject. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the safe therapeutic dose is equal to or less than about 300 mg. In a further embodiment, the safe therapeutic dose is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

An embodiment of the invention is a method of selecting a safe therapeutic dose or of monitoring the safe use of a therapeutic dose of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 to a human subject, said method comprising (a) administering a dose of said dual V region antibody-like protein or a fragment of a dual V region antibody-like region to said human subject; (b) measuring a ventricular depolarization and repolariztion time (QT) on an electrocardiogram (ECG) machine wherein the QT is automatically corrected by the ECG machine (QTc) of said human subject; and (c) determining said QTC is less than 500 ms as measured in (b) wherein said dose is selected as said safe therapeutic dose to be administered to said human subject. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the safe therapeutic dose is equal to or less than about 300 mg. In a further embodiment, the safe therapeutic dose is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

An embodiment of the invention is a method of determining whether a therapeutic dose of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region is safe and tolerable for administration to humans, the method comprising (a) perform a non-immunogenicity study in a non-human primate; and (b) determine a safe therapeutic dose in a human patient based on the non-immunogenicity study in the non-human primate. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the therapeutic dose is equal to or less than about 300 mg. In a further embodiment, the therapeutic dose is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

An embodiment of the invention is a method of measuring total amount of human antibody in a test sample, the method comprising (a) providing a monoclonal anti-human kappa chain; (b) adding a test sample to the monoclonal anti-human kappa chain; (c) adding sulfo-tag labeled anti-human antibody to the monoclonal anti-human kappa chain and the sample; and (d) quantifying the amount of the tag-labeled anti-human antibody that is bound to the sample wherein the amount of the tag-labeled anti-human antibody bound to the test sample determines the total amount of human antibody in the test sample. In further embodiment of the invention, the anti-human kappa chain is attached to a capture device.

An embodiment of the invention is a method of measuring a proportion of bispecific antibody capable of binding IL-4 and IL13 present in a test sample, the method comprising (a) providing a anti-human IL-4 antibody; (b) adding human IL-4 to the anti-human IL-4 antibody; (c) adding a test sample comprising a bispecific antibody capable of binding IL-4 and IL-13 to the human IL-4 and the anti-human IL-4 antibody; (d) adding human IL-13 to the test sample comprising a bispecific antibody capable of binding IL-4 and IL-13 and the human IL-4 and the anti-human IL-4 antibody; (e) adding biotinylated anti-human IL-13 antibody to the test sample comprising a bispecific antibody capable of binding IL-4 and IL-13 and the human IL-4 and the anti-human IL-4 antibody; and (f) adding tag-labeled streptavidin to the biotinylated anti-human IL-13 antibody and the test sample comprising a bispecific antibody capable of binding IL-4 and IL-13 and the human IL-4 and the anti-human IL-4 antibody; and (g) quantifying the amount of the tag-labeled streptavidin that is bound to the biotinylated anti-human IL-13 antibody wherein the amount of the tag-labeled streptavidin bound determines the proportion of bispecific antibody capable of binding IL-4 and IL3 present in the test sample. In a further embodiment of the invention, the anti-human IL-4 antibody is attached to a capture device. In a further embodiment of the invention, the bispecific antibody comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6.

An embodiment of the invention is a method of measuring a proportion of bispecific antibody capable of binding IL-4 and IL13 present in a test sample, the method comprising: (a) providing a anti-human IL-13 antibody; (b) adding human IL-13 to the anti-human IL-13 antibody; (c) adding a test sample comprising a bispecific antibody capable of binding IL-4 and IL-13 to the human IL-13 and the anti-human IL-13 antibody; (d) adding human IL-4 to the test sample comprising a bispecific antibody capable of binding IL-4 and IL-13 and the human IL-13 and the anti-human IL-13 antibody; (e) adding biotinylated anti-human IL-4 antibody to the test sample comprising a bispecific antibody capable of binding IL-4 and IL-13 and the human IL-13 and the anti-human IL-13 antibody; and (f) adding tag-labeled streptavidin to the biotinylated anti-human IL-4 antibody and the test sample comprising a bispecific antibody capable of binding IL-4 and IL-13 and the human IL-13 and the anti-human IL-13 antibody; and (g) quantifying the amount of the tag-labeled streptavidin that is bound to biotinylated anti-human IL-4 antibody wherein the amount of the tag-labeled streptavidin bound determines the proportion of bispecific antibody capable of binding IL-4 and IL13 present in the test sample. In a further embodiment of the invention, the anti-human IL-13 antibody is attached to a capture device. In a further embodiment of the invention, the bispecific antibody comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6.

An embodiment of the invention is a method of measuring anti-drug antibodies in a test sample, the method comprising (a) combining a test sample with a biotinylated bispecific antibody capable of binding IL-4 and IL-13 and a tag-labeled bispecific antibody capable of binding IL-4 and IL-13; (b) adding streptavidin to the test sample and the biotinylated bispecific antibody capable of binding IL-4 and IL-13 and the tag-labeled bispecific antibody capable of binding IL-4 and IL-13; and (c) quantifying the amount of the tag-labeled bispecific antibody capable of binding IL-4 and IL-13 is bound wherein the amount of the tag-labeled bispecific antibody capable of binding IL-4 and IL-13 bound determines the amount of anti-drug antibodies human antibody in the test sample. In a further embodiment of the invention, the streptavidin is attached to a capture device.

An embodiment of the invention is a method of quantifying or monitoring an amount of anti-drug antibodies in blood serum of a human subject or a non-human primate following administration of drug wherein the drug is a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13, said method comprising: (a) administering a dose of said dual V region antibody-like protein or a fragment of a dual V region antibody-like region to said human subject or said non-human primate; (b) obtaining a sample of said blood serum from said human subject or said non-human primate; and (b) determining the amount of anti-drug antibodies in said serum sample. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6.

An embodiment of the invention is a method of quantifying or monitoring a total amount of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 in blood serum of a human subject or a non-human primate, said method comprising (a) administering a dose of said dual V region antibody-like protein or a fragment of a dual V region antibody-like region to said human subject or said non-human primate; (b) obtaining a sample of said blood serum from said human subject or said non-human primate; and (c) determining said total amount of said dual V region antibody-like protein or a fragment of a dual V region antibody-like region in said sample. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6.

An embodiment of the invention is a method of quantifying or monitoring a proportion of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 that is functionally available to bind IL-4 and IL-13 in blood serum of a human subject or a non-human primate, said method comprising (a) administering a dose of said dual V region antibody-like protein or a fragment of a dual V region antibody-like region to said human subject or said non-human primate; (b) obtaining a sample of said blood serum from said human subject or said non-human primate; and (c) determining said proportion of said dual V region antibody-like protein or a fragment of a dual V region antibody-like region that is functionally available to bind IL-4 and IL-13 in said sample. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6.

An embodiment of the invention is a method of treating asthma in a mammal comprising the step of administering to said mammal a therapeutically effective amount of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the therapeutically effective amount is equal to or less than about 300 mg. In a further embodiment, the therapeutically effective amount is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

An embodiment of the invention is a method of treating idiopathic pulmonary fibrosis in a mammal comprising the step of administering to said mammal a therapeutically effective amount of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the therapeutically effective amount is equal to or less than about 300 mg. In a further embodiment, the therapeutically effective amount is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

An embodiment of the invention is a method of treating a disease mediated by IL-4 or IL-13 or IL-4 and IL-13-induced STAT6 phosphorylation in a mammal which comprises administering a therapeutically effective amount of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the therapeutically effective amount is equal to or less than about 300 mg. In a further embodiment, the therapeutically effective amount is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

An embodiment of the invention is a method of treating a disease mediated by IL-4 or IL-13 or IL-4 and IL-13-induced IL-6 release in a mammal which comprises administering a therapeutically effective amount of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the therapeutically effective amount is equal to or less than about 300 mg. In a further embodiment, the therapeutically effective amount is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

An embodiment of the invention is a method of treating a disease mediated by IL-4 or IL-13 or IL-4 and IL-13-induced eotaxin release in a mammal which comprises administering a therapeutically effective amount of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the therapeutically effective amount is equal to or less than about 300 mg. In a further embodiment, the therapeutically effective amount is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

An embodiment of the invention is a method of treating a disease mediated by IL-4 or IL-13 or IL-4 and IL-13-induced LOX expression in a mammal which comprises administering a therapeutically effective amount of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the therapeutically effective amount is equal to or less than about 300 mg. In a further embodiment, the therapeutically effective amount is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

An embodiment of the invention is a method of treating a disease mediated by IL-4 or IL-13 or IL-4 and IL-13-induced erythrocyte proliferation in a mammal which comprises administering a therapeutically effective amount of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13. In a further embodiment of the invention, the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO: 4. In a further embodiment, SEQ ID NO:1 and SEQ ID NO:3 are linked together with a peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker. In a further embodiment, the peptide linker consists of SEQ ID NO: 6. In another embodiment, the therapeutically effective amount is equal to or less than about 300 mg. In a further embodiment, the therapeutically effective amount is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effects of huTBTI3_2_1 on IL-4-induced or IL-13-induced Stat6 phosphorylation in IL-4- or IL-13-stimulated monocytes.

FIG. 2 shows the effects of huTBTI3_2_1 on IL-4 and IL-13-stimulated IL-6 and eotaxin release from human lung fibroblasts from a idiopathic pulmonary fibrosis patient.

FIG. 3 shows the effects of huTBTI3_2_1 on IL-4 and IL-13 induced LOX expression in idiopathic pulmonary fibrosis pulmonary fibroblasts.

FIG. 4 shows the effects of huTBTI3_2_1 on antigen-induced airway hyperresponsiveness against allergen induced acute asthma in cynomolgus monkeys. Data are represented as mean±s.e.m. *p<0.05, **p<0.01 compared with control antibody-treated group.

FIG. 5 shows the effects of huTBTI3_2_1 on antigen-induced accumulation of total leukocytes in airways against allergen induced acute asthma in cynomolgus monkeys. Data are represented as mean±s.e.m. *p<0.05, **p<0.01 compared with control antibody-treated group.

FIG. 6 shows the effects of huTBTI3_2_1 on antigen-induced accumulation of eosinophils in airways against allergen induced acute asthma in cynomolgous monkeys. Data are represented as mean±s.e.m. *p<0.05, **p<0.01 compared with control antibody-treated group.

FIG. 7 shows the effects of huTBTI3_2_1 on serum IgE titer from allergen induced acute asthma in cynomolgous monkeys. Data are represented as mean±s.e.m. *p<0.05, **p<0.01 compared with control antibody-treated group.

FIG. 8 shows the effects of SAR156597 on IL-4 and IL-13 mediated TGFβ release from normal human bronchial epithelial cells (NHBE; left panel) and human small airway epithelial cells (SAEC; right panel).

FIG. 9 shows a diagramatic representation of assays to measure total amount of human antibody in serum by measuring the concentration of human light chain K (panel A); to measure the proportion of functional antibodies to IL-4 and IL-13 (panel B); and to measure anti-drug antibodies (ADA) (panel C).

FIG. 10 shows a time plot (panel A) representation of the mean serum concentration of functional huTBTI3_2_1 after single dose intravenous perfusion administration of 2.5 mg/kg of huTBTI3_2_1 (Batch #LP08045) in PBS to a Cynomolgus Monkey at day 0, 14, 21 28 and 35; the table (panel B) summarizes pharmacokinetic parameters after the 1^(st) dose and the 5^(th) dose of 2.5 mg/kg of huTBTI3_2_1 (Batch #LP08059) in PBS in monkeys.

FIG. 11 shows the mean of SAR156597 plasma concentrations after a single subcutaneous dose from 10 to 300 mg from TDU11325.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety to the extent that it is not inconsistent with this present disclosure.

It is noted here that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

Furthermore, in accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

The following non-limiting definitions of some terms and phrases are provided to guide the artisan.

“Interleukin-4” (IL-4) relates to the naturally occurring, or endogenous mammalian IL-4 proteins and to proteins having an amino acid sequence which is the same as that of a naturally occurring or endogenous corresponding mammalian IL-4 protein {e.g., recombinant proteins, synthetic proteins (i.e., produced using the methods of synthetic organic chemistry)). Accordingly, as defined herein, the term includes mature IL-4 protein, polymorphic or allelic variants, and other isoforms of an IL-4 and modified or unmodified forms of the foregoing (e.g., lipidated, glycosylated). Naturally occurring or endogenous IL-4 includes wild type proteins such as mature IL-4, polymorphic or allelic variants and other isoforms and mutant forms which occur naturally in mammals (e.g., humans, non-human primates). Such proteins can be recovered or isolated from a source which naturally produces IL-4, for example. These proteins and proteins having the same amino acid sequence as a naturally occurring or endogenous corresponding IL-4, arc referred to by the name of the corresponding mammal. For example, where the corresponding mammal is a human, the protein is designated as a human IL-4. Several mutant IL-4 proteins are known in the art, such as those disclosed in WO 03/038041.

“Interleukin-13” (IL-13) refers to naturally occurring or endogenous mammalian IL-13 proteins and to proteins having an amino acid sequence which is the same as that of a naturally occurring or endogenous corresponding mammalian IL-13 protein (e.g., recombinant proteins, synthetic proteins (i.e., produced using the methods of synthetic organic chemistry)). Accordingly, as defined herein, the term includes mature IL-13 protein, polymorphic or allelic variants, and other isoforms of IL-13 (e.g., produced by alternative splicing or other cellular processes), and modified or unmodified forms of the foregoing (e.g., Hpidated, glycosylated). Naturally occurring or endogenous IL-13 include wild type proteins such as mature IL-13, polymorphic or allelic variants and other isoforms and mutant forms which occur naturally in mammals (e.g., humans, non-human primates). For example, as used herein IL-13 encompasses the human IL-13 variant in which Arg at position 110 of mature human IL-13 is replaced with Gin (position 110 of mature IL-13 corresponds to position 130 of the precursor protein) which is associated with asthma (atopic and nonatopic asthma) and other variants of IL-13. (Heinzmann el al, Hum Mol Genet. 9:549-559 (2000).) Such proteins can be recovered or isolated from a source which naturally produces IL-13, for example. These proteins and proteins having the same amino acid sequence as a naturally occurring or endogenous corresponding IL-13 are referred to by the name of the corresponding mammal. For example, where the corresponding mammal is a human, the protein is designated as a human IL-13. Several mutant IL-13 proteins are known in the art, such as those disclosed in WO 03/035847.

The phrase “substantially identical” with respect to an antibody chain polypeptide sequence may be construed as an antibody chain exhibiting at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference polypeptide sequence. The term with respect to a nucleic acid sequence may be construed as a sequence of nucleotides exhibiting at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference nucleic acid sequence. Identity can be determined by using any bioinformatics tool available to one skilled in the art. For example, Basic Local Alignment Search Tool (BLAST) is commonly employed to determine sequence identity (Altschul et al., Journal of Molecular Biology 215(3):403-410, 1990).

The terms, “identity” or “homology” may mean the percentage of nucleotide bases or amino acid residues in the candidate sequence that are identical with the residue of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequence identity. Neither N-terminal or C-terminal extensions nor insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are available and well known in the art. Sequence identity may be measured using sequence analysis software.

The phrases and terms “functional fragment, variant, derivative or analog” and the like, as well as forms thereof, of an antibody or antigen is a compound or molecule having qualitative biological activity in common with a full-length antibody or antigen of interest. For example, a functional fragment or analog of an anti-IL-4 antibody is one which can bind to an IL-4 molecule or one which can prevent or substantially reduce the ability of a ligand, or an agonistic or antagonistic antibody, to bind to IL-4.

“Substitutional” variants are those that have at least one amino acid residue in a native sequence removed and replaced with a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule is substituted, or may be multiple, where two or more amino acids are substituted in the same molecule. The plural substitutions may be at consecutive sites. Also, one amino acid can be replaced with plural residues, in which case such a variant comprises both a substitution and an insertion. “Insertional” variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native sequence. Immediately adjacent to an amino acid means connected to either the α-carboxyl or α-amino functional group of the amino acid. “Deletional” variants are those with one or more amino acids in the native amino acid sequence removed. Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the molecule.

The term “antibody” is used in the broadest sense, and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments or synthetic polypeptides carrying one or more CDR or CDR-derived sequences so long as the polypeptides exhibit the desired biological activity. Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. Generally, antibodies are considered Igs with a defined or recognized specificity. Thus, while antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity. The antibodies of the invention can be of any class (e.g., IgG, IgE, IgM, IgD, IgA and so on), or subclass (e.g., IgG₁, IgG₂, IgG_(2a), IgG₃, IgG₄, IgA₁, IgA₂ and so on) (“type” and “class”, and “subtype” and ““subclass”, are used interchangeably herein). Native or wildtype, that is, obtained from a non-artificially manipulated member of a population, antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has at one end a variable domain (V_(H)) followed by a number of constant domains. Each light chain has a variable domain at one end (V_(L)) and a constant domain at the other end. By “non-artificially manipulated” is meant not treated to contain or express a foreign antigen binding molecule. Wildtype can refer to the most prevalent allele or species found in a population or to the antibody obtained from a non-manipulated animal, as compared to an allele or polymorphism, or a variant or derivative obtained by a form of manipulation, such as mutagenesis, use of recombinant methods and so on to change an amino acid of the antigen-binding molecule.

As used herein, “anti-IL-4 antibody” means an antibody or polypeptide derived therefrom (a derivative) which binds specifically to IL-4 as defined herein, including, but not limited to, molecules which inhibit or substantially reduce the binding of IL-4 to its receptor or inhibit IL-4 activity.

As used herein, “anti-IL-13 antibody” means an antibody or polypeptide derived therefrom (a derivative) which binds specifically to IL-13 as defined herein, including, but not limited to, molecules which inhibit or substantially reduce the binding of IL-13 to its receptor or inhibit IL-13 activity.

The term “variable” in the context of a variable domain of antibodies refers to certain portions of the pertinent molecule which differ extensively in sequence between and among antibodies and are used in the specific recognition and binding of a particular antibody for its particular target. However, the variability is not evenly distributed through the variable domains of antibodies. The variability is concentrated in three segments called complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) also known as hypervariable regions, both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework (FR) regions or sequences. The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a 3-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together often in proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the target (epitope or determinant) binding site of antibodies (see Kabat et al. Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md. (1987)). As used herein, numbering of immunoglobulin amino acid residues is done according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated. One CDR can carry the ability to bind specifically to the cognate epitope.

The term “hinge” or “hinge region” as used in the present invention refers to the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody.

The term “antibody fragment” refers to a portion of an intact or a full-length chain or an antibody, generally the target binding or variable region. Examples of antibody fragments include, but are not limited to, F_(ab), F_(ab′), F_((ab′)2) and F_(v) fragments. A “functional fragment” or “analog of an anti-IL-4 and/or IL-13 antibody” is one which can prevent or substantially reduce the ability of the receptor to bind to a ligand or to initiate signaling. As used herein, functional fragment generally is synonymous with, “antibody fragment” and with respect to antibodies, can refer to fragments, such as F_(v), F_(ab), F_((ab′)2) and so on which can prevent or substantially reduce the ability of the receptor to bind to a ligand or to initiate signaling. An “F_(v)” fragment consists of a dimer of one heavy and one light chain variable domain in a non-covalent association (V_(H)-V_(L) dimer). In that configuration, the three CDRs of each variable domain interact to define a target binding site on the surface of the V_(H)-V_(L) dimer, as in an intact antibody. Collectively, the six CDRs confer target binding specificity on the intact antibody. However, even a single variable domain (or half of an F_(v) comprising only three CDRs specific for a target) can have the ability to recognize and to bind target.

“Single-chain F_(v),” “sF_(v)” or “scAb” antibody fragments comprise the V_(H) and V_(L) domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the F polypeptide further comprises a polypeptide linker, often a flexible molecule, between the V_(H) and V_(L) domains, which enables the sFv to form the desired structure for target binding.

The term “diabodies” refers to antibody fragments with two antigen-binding sites, which fragments can comprise a heavy chain variable domain (V_(H)) connected to a light chain variable domain (V_(L)) in the same polypeptide chain. By using a linker that is too short to allow pairing between the two variable domains on the same chain, the diabody domains are forced to pair with the binding domains of another chain to create two antigen-binding sites.

The F_(ab) fragment contains the variable and constant domains of the light chain and the variable and first constant domain (C_(H)1) of the heavy chain. F_(ab′) fragments differ from F_(ab) fragments by the addition of a few residues at the carboxyl terminus of the C_(H1) domain to include one or more cysteines from the antibody hinge region. F_(ab′) fragments can be produced by cleavage of the disulfide bond at the hinge cysteines of the F_((ab′)2) pepsin digestion product. Additional enzymatic and chemical treatments of antibodies can yield other functional fragments of interest.

The term “linear Fab” refers to a tetravalent antibody as described by Miller et al. (2003), J Immunol. 170: 4854-4861. The “linear Fab” is composed of a tandem of the same CH1-VH domain, paired with the identical light chain at each CH1-VH position. These molecules have been developed in order to increase the valency of an antibody to enhance its functional affinity through the avidity effect, but they are monospecific.

The term “bispecific antibodies (BsAbs)” refers to molecules which combine the antigen-binding sites of two antibodies within a single molecule. Thus, a bispecific antibody is able to bind two different antigens simultaneously. Besides applications for diagnostic purposes, BsAbs pave the way for new therapeutic applications by redirecting potent effector systems to diseased areas or by increasing neutralizing or stimulating activities of antibodies.

Monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass (type or subtype), with the remainder of the chain(s) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity of binding to IL-4 and/or IL-13 or impacting IL-4 and/or IL-13 activity or metabolism (U.S. Pat. No. 4,816,567; and Morrison et al., Proc Natl Acad Sci USA 81:6851 (1984)). Thus, CDRs from one class of antibody can be grafted into the FR of an antibody of different class or subclass.

Monoclonal antibodies are highly specific, being directed against a single target site, epitope or determinant. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes) of an antigen, each monoclonal antibody is directed against a single determinant on the target. In addition to their specificity, monoclonal antibodies are advantageous being synthesized by a host cell, uncontaminated by other immunoglobulins, and provides for cloning the relevant gene and mRNA encoding the antibody of chains thereof. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies for use with the present invention may be isolated from phage antibody libraries using well known techniques or can be purified from a polyclonal preparation. The parent monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant methods well known in the art.

The term “polyvalent antibody” as used in the present invention refers to an antibody comprising two or more antigen binding sites, thus being able to bind two or more antigens, which may have the same or a different structure, simultaneously. The term “bivalent” means that the antibody comprises two antigen binding sites. The term “tetravalent” means that the antibody comprises four antigen binding sites.

The term “antigen binding site” as used in the present invention refers to the part of the antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed on epitope. An antigen binding domain may be provided by one or more antibody variable domains. Preferably, an antigen binding domain is made of the association of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

The term “antigen” as used in the present invention refers to a molecule or a portion of a molecule capable of being bound by the antibodies of the present invention. An antigen can have one or more than one epitope. Examples of antigens recognized by the antibodies of the present invention include, but are not limited to, serum proteins, e.g. cytokines such as IL-4, IL-5, IL-9 and IL-13, bioactive peptides, cell surface molecules, e.g. receptors, transporters, ion-channels, viral and bacterial proteins.

The term “monospecific” as used in the present invention means that the polyvalent antibody of the present invention recognizes only one antigen, all the antigen binding sites being identical.

The term “bispecific” as used in the present invention means that the polyvalent antibody of the present invention recognizes two different epitopes on the same or on two different antigens.

It has been of interest to produce bispecific antibodies (BsAbs) which combine the antigen-binding sites of two antibodies within a single molecule. Thus, such a molecule would be able to bind two different antigens simultaneously. Besides applications for diagnostic purposes, they pave the way for new therapeutic applications, e.g. by redirecting potent effector systems to diseased areas (where cancerous cells often develop mechanisms to suppress normal immune responses triggered by monoclonal antibodies, like antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC)), or by increasing neutralizing or stimulating activities of antibodies. Initial attempts to couple the binding specificities of two whole antibodies against different target antigens for therapeutic purposes utilized chemically fused heteroconjugate molecules (Staerz et al. (1985), Nature 314: 628-631).

Bispecific antibodies were originally made by fusing two hybridomas, each capable of producing a different immunoglobulin (Milstein and Cuello, 1983, 1984), but the complexity of species (up to ten different species) produced in cell culture makes purification difficult and expensive (George and Huston, 1997). Despite the promising results obtained using heteroconjugates or bispecific antibodies produced from cell fusions as cited above, several factors made them impractical for large scale therapeutic applications. Such factors include: rapid clearance of heteroconjugates in vivo, the laboratory intensive techniques required for generating either type of molecule, the need for extensive purification of heteroconjugates away from homoconjugates or mono-specific antibodies and generally low yields.

Genetic engineering has been used with increasing frequency to design, modify, and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions. A variety of recombinant methods have been developed for efficient production of BsAbs, both as antibody fragments (Carter et al. (1995), J. Hematotherapy 4: 463-470; Pluckthun et al. (1997) Immunotechology 3: 83-105; Todorovska et al. (2001) J. Immunol. Methods 248: 47-66) and full length IgG formats (Carter (2001) J. Immunol. Methods 248: 7-15).

Abbott described in US patent U.S. Pat. No. 7,612,181 a murine Dual-Variable-Domain IgG (DVD-IgG) bispecific antibody, which is based on the dual-Fv format described in Unilever patent (U.S. Pat. No. 5,989,830). A humanized bispecific format was described in WO2009/052081 (TBTI) which is incorporated herein by reference in its entirety. The addition of constant domains to respective chains of the Dual-Fv (CH1-Fc to the heavy chain and kappa or lambda constant domain to the light chain) led to functional bispecific dual V region antibody like binding proteins.

An embodiment of the invention is a bispecific antibody that has been engineered to comprise a dual V region antibody-like protein or fragment thereof that specifically binds to two different epitopes on the same or on two different antigens. An embodiment of the invention a bispecific antibody or bispecific antibody fragment thereof that specifically binds to IL-13 and IL-4, wherein said bispecific antibody or bispecific antibody fragment thereof comprises a variable light chain domain and a variable heavy chain domain, wherein said variable light chain domain comprises amino acid sequences SEQ ID NO:1 and SEQ ID NO:3. A further embodiment of the invention is a bispecific antibody or bispecific antibody fragment thereof that specifically binds to IL-13 and IL-4, wherein said bispecific antibody or bispecific antibody fragment thereof comprises a variable light chain domain and a variable heavy chain domain, wherein said variable heavy chain domain comprises amino acid sequences SEQ ID NO:2 and SEQ ID NO:5. Another embodiment of the invention is a bispecific antibody or bispecific antibody fragment thereof that specifically binds to IL-13 and IL-4, wherein said bispecific antibody or bispecific antibody fragment thereof comprises a variable light chain domain and a variable heavy chain domain, wherein said variable heavy chain domain comprises amino acid sequences SEQ ID NO:2 and SEQ ID NO:4. An embodiment of the invention is a bispecific antibody or bispecific antibody fragment thereof that specifically binds to IL-13 and IL-4, wherein said bispecific antibody or bispecific antibody fragment thereof comprises a variable light chain domain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO:3, and a variable heavy chain domain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO:4. A further embodiment of the invention is a bispecific antibody or bispecific antibody fragment thereof that specifically binds to IL-13 and IL-4, wherein said bispecific antibody or bispecific antibody fragment thereof comprises a variable light chain domain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO:3, and a variable heavy chain domain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO:4, wherein a peptide linker links SEQ ID NO:1 to SEQ ID NO:3, and a peptide linker links SEQ ID NO:2 to SEQ ID NO:4. An embodiment of the invention is huTBTI3_2_1 or SAR156597 comprising a bispecific antibody or bispecific antibody fragment thereof that specifically binds to IL-13 and IL-4, comprising (a) variable light chain domain comprising the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:3; (b) a variable heavy chain domain comprising the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:4; (c) a peptide linker linking SEQ ID NO:1 to SEQ ID NO:3, and a peptide linker linking SEQ ID NO:2 to SEQ ID NO:4 wherein the peptide linker has an amino acid sequence consisting of SEQ ID NO:6; and (d) constant region domains.

The term “multispecific” as used in the present invention means that the polyvalent antibody of the present invention recognizes multiple different epitopes on the same or on multiple different antigens.

The term “linker” as used in the present invention refers to a peptide adapted to connect the variable domains of the antibody constructs of the present invention. The peptide linker may contain any amino acids, the amino acids glycine (G) and serine (S) being preferred. The linkers may be equal or differ from each other between and within the heavy chain polypeptide and the light chain polypeptide. Furthermore, the linker may have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. A preferred peptide linker unit for the heavy chain domains as for the light chain domains is GGGGS. The numbers of linker units of the heavy chain and of the light chain may be equal (symmetrical order) or differ from each other (asymmetrical order).

A peptide linker is preferably long enough to provide an adequate degree of flexibility to prevent the antibody moieties from interfering with each others activity, for example by steric hindrance, to allow for proper protein folding and, if necessary, to allow the antibody molecules to interact with two or more, possibly widely spaced, receptors on the same cell; yet it is preferably short enough to allow the antibody moieties to remain stable in the cell.

Therefore, the length, composition and/or conformation of the peptide linkers can readily be selected by one skilled in the art in order to optimize the desired properties of the polyvalent antibody.

“Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as F_(v), F_(b), F_(ab), F_((ab′)2) or other target-binding subsequences of antibodies) which contain sequences derived from non-human immunoglobulin, as compared to a human antibody. In general, the humanized antibody will comprise substantially all of one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin template sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (F_(c)), typically that of the human immunoglobulin template chosen. In general, the goal is to have an antibody molecule that is minimally immunogenic in a human. Thus, it is possible that one or more amino acids in one or more CDRs also can be changed to one that is less immunogenic to a human host, without substantially minimizing the specific binding function of the one or more CDRs to IL-4 and/or IL-13. Alternatively, the FR can be non-human but those amino acids most immunogenic are replaced with ones less immunogenic. Nevertheless, CDR grafting, as discussed above, is not the only way to obtain a humanized antibody. For example, modifying just the CDR regions may be insufficient as it is not uncommon for framework residues to have a role in determining the three-dimensional structure of the CDR loops and the overall affinity of the antibody for its ligand. Hence, any means can be practiced so that the non-human parent antibody molecule is modified to be one that is less immunogenic to a human, and global sequence identity with a human antibody is not always a necessity. So, humanization also can be achieved, for example, by the mere substitution of just a few residues, particularly those which are exposed on the antibody molecule and not buried within the molecule, and hence, not readily accessible to the host immune system. Such a method is taught herein with respect to substituting “mobile” or “flexible” residues on the antibody molecule, the goal being to reduce or dampen the immunogenicity of the resultant molecule without comprising the specificity of the antibody for its epitope or determinant. See, for example, Studnicka et al., Prot Eng 7(6)805-814, 1994; Mol Imm 44:1986-1988, 2007; Sims et al., J Immunol 151:2296 (1993); Chothia et al., J Mol Biol 196:901 (1987); Carter et al., Proc Natl Acad Sci USA 89:4285 (1992); Presta et al., J Immunol 151:2623 (1993), WO 2006/042333 and U.S. Pat. No. 5,869,619.

“Antibody homolog” or “homolog” refers to any molecule which specifically binds IL-4 and/or IL-13 as taught herein. Thus, an antibody homolog includes native or recombinant antibody, whether modified or not, portions of antibodies that retain the biological properties of interest, such as binding IL-4 or IL-13, such as an F_(ab) or F_(v) molecule, a single chain antibody, a polypeptide carrying one or more CDR regions and so on. The amino acid sequence of the homolog need not be identical to that of the naturally occurring antibody but can be altered or modified to carry substitute amino acids, inserted amino acids, deleted amino acids, amino acids other than the twenty normally found in proteins and so on to obtain a polypeptide with enhanced or other beneficial properties.

Antibodies with homologous sequences are those antibodies with amino acid sequences that have sequence homology with the amino acid sequence of a IL-4, IL-13 or bispecific IL-4/IL-13 antibody of the present invention. Preferably, homology is with the amino acid sequence of the variable regions of an antibody of the present invention. “Sequence homology” as applied to an amino acid sequence herein is defined as a sequence with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to another amino acid sequence, as determined, for example, by the FASTA search method in accordance with Pearson & Lipman, Proc Natl Acad Sci USA 85, 2444-2448 (1988).

A chimeric antibody is one with different portions of an antibody derived from different sources, such as different antibodies, different classes of antibody, different animal species, for example, an antibody having a variable region derived from a murine monoclonal antibody paired with a human immunoglobulin constant region and so on. Thus, a humanized antibody is a species of chimeric antibody. Methods for producing chimeric antibodies are known in the art, see, e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J Immunol Methods 125:191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567, and 4,816,397.

Artificial antibodies include scFv fragments, chimeric antibodies, diabodies, triabodies, tetrabodies and mru (see reviews by Winter & Milstein, 1991, Nature 349:293-299; and Hudson, 1999, Curr Opin Imm 11:548-557), each with antigen-binding or epitope-binding ability. In the single chain F fragment (scF_(v)), the V_(H) and V_(L) domains of an antibody are linked by a flexible peptide. Typically, the linker is a peptide of about 15 amino acids. If the linker is much smaller, for example, 5 amino acids, diabodies are formed. The smallest binding unit of an antibody is a CDR, typically the CDR2 of the heavy chain which has sufficient specific recognition and binding capacity. Such a fragment is called a molecular recognition unit or mru. Several such mrus can be linked together with short linker peptides, therefore forming an artificial binding protein with higher avidity than a single mru.

Also included within the scope of the invention are functional equivalents of an antibody of interest. The term “functional equivalents” includes antibodies with homologous sequences, antibody homologs, chimeric antibodies, artificial antibodies and modified antibodies, for example, wherein each functional equivalent is defined by the ability to bind to IL-4 and/or IL-13, inhibiting IL-4 and/or IL-13 signaling ability or function, or inhibiting binding of IL-4 and/or IL-13 to its receptor. The skilled artisan will understand that there is an overlap in the group of molecules termed “antibody fragments” and the group termed “functional equivalents.” Methods of producing functional equivalents which retain IL-4 and/or IL-13 binding ability are known to the person skilled in the art and are disclosed, for example, in WO 93/21319, EPO Ser. No. 239,400, WO 89/09622, EPO Ser. No. 338,745 and EPO Ser. No. 332,424.

The functional equivalents of the present application also include modified antibodies, e.g., antibodies modified by the covalent attachment of any type of molecule to the antibody. For example, modified antibodies include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, deamidation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand, linkage to a toxin or cytotoxic moiety or other protein etc. The covalent attachment need not yield an antibody that is immune from generating an anti-idiotypic response. The modifications may be achieved by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis etc. Additionally, the modified antibodies may contain one or more non-classical amino acids.

“Mammal” for purposes of treatment refers to any animal classified as a mammal, including human, domestic and farm animals, nonhuman primates, and zoo, sports or pet animals, such as dogs, horses, cats, cows etc.

The term “treatment”, “therapeutic dose” or “administering a therapeutically effective amount” as used in the present invention refers to both therapeutic treatment and prophylactic or preventative measures as a course of therapy. It refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting deleterious effects of a disease state, disease progression, disease causative agent (e.g., bacteria or viruses) or other abnormal condition.

An embodiment of the invention is the treatment of asthma and idiopathic pulmonary fibrosis. IL-4 and IL-13 are therapeutically important cytokines based on their biological functions and play critical roles in many diseases, including asthma (Curr Opin Allergy Clin Immunol 2005, Vo. 5, 161-166). IL-4 has been shown to be able to inhibit autoimmune disease and IL-4 and IL-13 have both shown the potential to enhance anti-tumor immune responses. Elevations in IL-4 and IL-13 and their receptors have been linked to the pathogenesis of idiopathic pulmonary fibrosis (IPF) (Jakubzick C. et al., Am J Pathol. 2004:164(6):1989-2001; Murray L A et al. Int J Biochem Cell Biol. 2008:40(10):2174-82. Evidence in the literature demonstrate that the TH2 cytokines IL-4 and IL-13 play multiple roles in the pathogenesis of IPF as mediators of this lung tissue remodeling and fibrosis (Wynn, T A, Nat. Rev. Immunol, 4:583-594, 2004) and other cell types including mast cells, basophils, eosinophils, macrophages and epithelial cells may also be potential sources of these cytokines (Gordon S and Martinez F O, Immunity Rev. 32:593-604, 2010). In IPF patients, IL-13 and IL-4 levels in bronchial alveolar lavage fluid are elevated compared to normal controls. Such evidence suggests that therapies capable of suppressing or neutralizing these cytokines have the potential for delaying the progression of fibrosis in IPF patients. Since both cytokines are involved in the pathogenesis of allergic diseases or fibrotic diseases, inhibitors of these cytokines could provide therapeutic benefits.

An “isolated” or “purified” antibody is substantially free of cellular material or other contaminating proteins from the cell or tissue source or medium from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of an antibody in which the polypeptide/protein is separated from cellular components of the cells from which same is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes preparations of the antibody having less than about 30%, 20%, 10%, 5%, 2.5% or 1%, (by dry weight) of contaminating protein. When the antibody is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, 5%, 2.5% or 1% of the volume of the protein preparation. When antibody is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals and reagents, i.e., the antibody of interest is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly, such preparations of the antibody have less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of chemical precursors or compounds other than antibody of interest. In a preferred embodiment of the present invention, antibodies are isolated or purified.

As used herein, the terms “therapeutic agent” and “therapeutic agents” refer to any agent(s) which can be used in the treatment, management or amelioration of a disease, disorder, malady and the like associated with aberrant IL-4 and/or IL-13 metabolism and activity.

As used herein, “therapeutic dose” refers to the quantity of any agent(s) which can be used in the treatment, management or amelioration of a disease, disorder, malady and the like associated with aberrant IL-4 and/or IL-13 metabolism and activity.

As used herein, “safe therapeutic dose” refers to any agent(s) or dose of any agent(s) which can be used in the treatment, management or amelioration of a disease, disorder, malady and the like associated with aberrant IL-4 and/or IL-13 metabolism and activity while maintaining a clinically acceptable benefit/risk profile. A safe therapeutic dose is selected form the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg. An embodiment of a safe therapeutic dose is about 10 mg to about 300 mg. A further embodiment of a safe therapeutic dose is any dose that is about 300 mg or less than about 300 mg.

An embodiment of the invention is identifying or monitoring a safe therapeutic dose by measuring one or more events selected from the group consisting of intensive treatment in an emergency room or at home for allergic bronchospasm, blood dyscrasias, convulsions, alanine aminotransferase (ALT) >3×upper limit of normal range (ULN) associated with total bilirubin >2×ULN, asymptomatic ALT increase >10×ULN, development of drug dependency or drug abuse, ALT increase ≥2×ULN, hsCRP >10 mg/L for ≥72 hours, cardiac troponin I (cTnI) >2×ULN, a ventricular depolarization and repolarization time (QT) on an electrocardiogram (ECG) machine wherein the QT is automatically corrected by the ECG machine (QTc) that is QTc ≥500 ms, severe skin reactions local to the site of IP injection and a level of C-reactive protein (CRP) is less than 20 mg/L. The methods used to calculate the afore-mentioned events are discussed in detail in the examples presented below. Methods used to calculate the afore-mentioned events are commonly know to those skilled in the art.

Intracellular signaling after ligation of IL-4 and IL-13 with their cell surface receptors is mediated in part by phosphorylation of the signaling molecule signal transducer and activator of transcription 6 (Stat6). Therefore, inhibition of Stat6 phosphorylation (pStat6) can be used to test the ability of a molecule to inhibit activation of the IL-4 and IL-13 receptors.

IL-4 and IL-13 stimulate the release of IL-6 and eotaxin from human idiopathic pulmonary fibrosis lung fibroblasts. Therefore, inhibition of IL-6 and eotaxin release can be used to test the ability of a molecule to inhibit activation of the IL-4 and IL-13 receptors.

An embodiment of the invention is an antibody that inhibits IL-4- or IL-13-induced STAT6 phophorylation, IL-6 release or eotaxin with an IC50 about 0.01 nM to about 100 nM. A further embodiment encompasses an IC50 about 0.1 nM to about 10 nM. A further embodiment encompasses an IC50 about 0.1 nM to about 10 nM. Additional embodiments of the invention are IC50 values about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2., 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0 nM.

The term “about” when used in connection with a numerical value is meant to encompass numerical values within a range having a lower limit that is 5%, 10% or 15% smaller than the indicated numerical value and having an upper limit that is 5%, 10% or 15% larger than the indicated numerical value.

An embodiment of the invention is detection methods to measure total human antibody levels or the proportion of a specific antibody (for example, a bispecific antibody) or to measure anti-drug antibodies in a test sample. The test sample can be any bodily sample from a mammal. Non-limiting examples include blood samples, serum samples or tissue samples. Detection methods may involve using a “capture device” in which one or more antibodies are attached to the capture device. Nonlimiting examples of “capture devices” include wells of a plate wherein the plate may include any number of wells such as a 12 well plate or a 96 well plate. However, capture devices are not limited to plates but may include any substrate that an antibody may attach to, for example, an elution column. An embodiment of the invention utilizes tag-labeled antibodies. The tag can be any tag capable of detection. Nonlimiting examples include fluorescent tags such as rhodamine, enzymatic tags such as luciferase or sulfo-tags.

EXAMPLES

The instant invention may be better understood by reference to the following non-limiting Examples, which are exemplary of the invention. The Examples presented below should in no way be construed as limiting the broad scope of the invention.

The terms “huTBTI3_2_1” and “SAR156597” are interchangeable and refer to the same dual V region antibody-like protein comprising a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO:3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO:4.

Example 1: Cloning and Generation of Humanized Anti-IL-4/IL-13 Bispecific Antibodies

The cloning and generation of humanized anti-IL-4/IL-13 bispecific antibodies is described in WO2009/052081 (PCT/US2008/079787), herein incorporated by reference in its entirety. For ease of reference, a brief description follows.

The format used for the expression of bispecific antibodies (BsAb) is an IgG variant of the dual domain double head format described in U.S. Pat. No. 5,989,830. In this format an IgG molecule is elongated at its N-terminus on the corresponding heavy and light chains, by an additional variable domain of a second antibody. Thus, the resulting IgG molecule is a heterotetramer composed of two heavy and two light chains. The heavy chains consist of two variables heavy domains (VH1-VH2) deriving from two different antibodies joined together by a linker composed of ten amino acids (G4S)₂ and fused to the IgG4 constant domain. The light chains consist of two variables light domains (VL1-VL2) deriving from two different antibodies joined together by a linker composed of ten amino acids (G4S)₂ and fused to the constant kappa region.

Sequences for the variable heavy and light domains of the 8D4-8 variants (8D4-8; mouse anti-IL-4 monoclonal antibody clone 8D4-8 from Biozol diagnostica Vertrieb GmbH, Eching Germany; Biozol is the German distributor of BioLegend, San Diego, Calif., USA) were generated by PCR introducing a BamHI restriction site (GGA TCC) at their respective 5′-ends encoding a part of the (G4S)₂-(GGA TCC)-8D4-8. The 3′ sequence of the VH of the 8D4-8 humanized variants ended with an ApaI restriction site (encoding the first amino acids of the CH1 domain) for a later fusion to the IGHG4 sequence (Q569F4, with deletion of the terminal Lys and a double mutation S241P and L248E). The 3′-end of the VL8D4-8 ended with a BsiWI restriction site encoding the first two amino acids of the constant kappa chain for a later fusion to IGKC (Gene Bank Accession Number Q502W4).

Sequences for the variable heavy and light domains of the B-B13 variants (B-B13; mouse anti-IL-13 monoclonal antibody clone B-B13 from Cell Sciences, Inc., Canton, Mass. USA) were generated by PCR introducing a BamHI restriction site at their respective 3′-ends encoding a part of the (G4S)₂-(B-B13)-(GGA GGC GGA GGG TCC GGA GGC GGA GGA TCC (SEQ ID NO: 7)). Both sequences for the VH and VL of the B-B13 variants were generated with a NheI restriction site at their respective 5′-ends, followed by an ATG start codon and a leader peptide encoding sequence.

The VH of B-B13 and 8D4-8 were fused together through their BamHI sites within the (G4S)₂ linker. The VL of B-B13 and 8D4-8 were fused to each other through their BamHI sites within the (G4S)₂ linker. Hence the tandems of heavy and the light chains generated had the following composition.

Bispecific antibody heavy chain: NheI-Leader peptide-VH-B-B13-(G4S)₂-VH 8D4-8-ApaI.

Bispecific antibody light chain: NheI-Leader peptide-VL-B-B13-(G4S)₂-VL 8D4-8-BsiWI.

All intermediate PCR fragments were cloned into the pCR®4-TOPO using the Invitrogen TOPO TA cloning kit (Cat #: 45-0641) and sequenced using M13forward and M13 reverse primers.

After sequence validation the heavy chain tandems were fused through their ApaI site to the IGHG4 sequence and the variable light chain tandems were fused through their BsiWI site to IGKC. The created dual domain heavy chain and light chain were digested with NheI and HindIII and each ligated into the NheI/HindIII sites of the episomal expression vector pXL, creating the plasmids for mammalian expression of the TBTI-heavy and light chains respectively.

Four humanized bispecific anti-IL-4/anti-IL-13 constructs were generated based on the following combinations of humanized VH and VL versions of B-B13 and 8D4-8 as shown in Table 1. The corresponding light and heavy chain sequences are shown in Table 2.

TABLE 1 Bispecific anti-IL-4/anti-IL-13 Antibodies Bispecific anti-IL-4/anti-IL-13 Ab Anti-IL-13 Fv Anti-IL-4 Fv huTBTI3_1_1 B-B13 VL3xVH2 8D4-8 VL1xVH2 huTBTI3_2_1 B-B13 VL3xVH2 8D4-8 VL1xVH1 huTBTI3_1_2 B-B13 VL2xVH2 8D4-8 VL1xVH2 huTBTI3_2_2 B-B13 VL2xVH2 8D4-8 VL1xVH1

TABLE 2 Sequences of Humanized Variable Domains and Linker Sequence Anti-IL13 hB-B13 VL3 (SEQ ID NO: 1): DIVLTQSPAS LAVSLGQRAT ISCRASESVD SYG Q SYMHWY QQKAGQPPKL LIYLASNLES GVPARFSGSG SRTDFTLTID PVQAEDAATY YCQQN A ED S R TFGGGTKLEI K Anti-IL13 hB-B13 VH2 (SEQ ID NO: 2): EVQLKESGPG LVAPGGSLSI TCTVSGFSLT DSSINWVRQP PGKGLEWLGM IWGDGRIDYA DALKSRLSIS KDSSKSQVFL EMTSLRTDDT ATYYCARDGY FPYAMDFWGQ GTSVTVSS Anti-IL4 h8D4-8 VL1 (SEQ ID NO: 3): DIQMTQSPAS LSVSVGDTIT LTCHASQNID VWLSWFQQKP GNIPKLLIYK ASNLHTGVPS RFSGSGSGTG FTLTISSLQP EDIATYYCQQ AHSYPFTFGG GTKLEIKR Anti-IL4 h8D4-8 VH1 (SEQ ID NO: 4): QVQLQQSGPE LVKPGASVKI SCKASGYSFT SYWIHWIKQR PGQGLEWIGM IDPSDGETRL NQRFQGRATL TVDESTSTAY MQLRSPTSED SAVYYCTRLK EYGNYDSFYF DVWGAGTLVT VSSA Anti-IL4 h8D4-8 VH2 (SEQ ID NO: 5): QVQLQQSGPE LVKPGASVKI SCKASGYSFT SYWIHWIKQR PGQGLEWIGM ID A SDGETRL NQRFQGRATL TVDESTSTAY MQLRSPTSED SAVYYCTRLK EYGNYDSFYF DVWGAGTLVT VSSA Linker Sequence (SEQ ID NO: 6) GGGGSGGGGS Underline indicates amino acid changes made for humanization or to remove residues subject to modification or acid lability. Bold indicates the CDR

Example 2: Effect of huTBTI3_2_1 on IL-4- or IL-13-induced STAT6 Phosphorylation in Human Whole Blood Monocytes

Human sodium citrate anti-coagulated whole blood was obtained from an on-site normal donor panel. Donor numbers 245, 217, 229 and 002 were used.

huTBTI3_2_1 was generated at sanofi-aventis, batch no. LP08059, supplied at 5.63 mg/ml in phosphate buffered saline (PBS) and stored at 4° C.

Recombinant human IL-13 (lyophilized) from R&D Systems, catalog no. 213-IL, was reconstituted with PBS containing 0.2% bovine serum albumin at 10 μg/ml. Final concentration of IL-13 used in the assay was 3 ng/ml in complete RPMI medium. Recombinant human IL-4 (lyophilized) from AMS Biotechnology LTD, catalog no. 111-40-134, was reconstituted with PBS containing 0.2% bovine serum albumin at 20 μg/ml. Final concentration of IL-4 used in the assay was 1 ng/ml in complete RPMI medium.

huTBTI3_2_1 was serial diluted with complete RPMI medium to make 10× solutions, and mixed with 100 μl of normal human peripheral blood per well in a 96-deep-well plate to reach final concentrations of huTBTI3_2_1 at 100 nM, 33.33 nM, 11.11 nM, 3.70 nM, 1.24 nM, 0.41 nM, 0.14 nM, 0.05 nM, 0.02 nM, and 0.005 nM for donor numbers 245, 217 and 002. For donor number 229, the final concentrations of huTBTI3_2_1 tested were 100 nM, 33.33 nM, 11.11 nM, 3.70 nM, 1.24 nM, 0.41 nM and 0.14 nM.

The plate was incubated in 37° C., 5% CO₂ for 15 to 30 minutes. Then recombinant human IL-4 (1 ng/ml) or IL-13 (3 ng/ml) were added to each well, and the plate was further incubated in 37° C., 5% CO₂ for 15 minutes. Blood cells were then lysed/fixed with lysis/fix buffer for 10 minutes at 37° C., centrifuged at 300×g for 5 minutes at room temperature. Supernatants were removed and the remaining cell pellet was washed once with phosphate-buffered saline. The cells were permeabilized with pre-cooled methanol for 30 minutes in 4° C. and then washed once with FACS stain buffer (BD, catalog no. 554656). Fluorescence-labeled antibodies (anti-phospho-Stat6-Alexa Fluor 647 at 1:5 final dilution, and anti-CD33-FITC at 1:10 final dilution) were added to cells and incubated at room temperature in the dark for 30 minutes. After wash with FACS stain buffer, cells were acquired through a FACS Calibur™ flow cytometer to generate FACS data.

FACS data were analyzed by using CellQuest Software™ (BD, version 5.2). Dot plots were created using CD33 staining (fluorescein isothiocyanate) versus pStat6 staining (Alexa Fluor 647) (see FIG. 1). Total monocytes were gated based on side scatter versus forward scatter. CD33⁺ staining, Stat6-phosphorylation positive monocytes were gated based on the fluorescence intensity between baseline control and IL-4 or IL-13 stimulated samples in the absence of huTBTI3_2_1. Percent of pStat6 positive monocytes among total monocytes were obtained from region statistics based on CellQuest software (BD, version 5.2).

The effects of huTBTI3_2_1 on IL-4- or IL-13-induced Stat6 phosphorylation were determined using percent inhibition of maximum response (Stat6 phosphorylation) in IL-4- or IL-13-stimulated monocytes.

The maximum response was defined as the percent of pStat6⁺ cells generated by IL-4 or IL-13 stimulation in the absence of huTBTI3_2_1. The percent of pStat6⁺ cells generated from unstimulated monocytes was used as baseline signal. The percent of maximum response was calculated using the following equation:

${{Percent}\mspace{14mu} (\%)\mspace{14mu} {of}\mspace{14mu} {maximum}\mspace{14mu} {response}} = {\frac{\begin{matrix} {{\% \mspace{14mu} {pStat}\; 6_{SAR156597}^{+}} -} \\ {\% \mspace{14mu} {pStat}\; 6_{baseline}^{1}} \end{matrix}}{\begin{matrix} {{\% \mspace{14mu} {pStat}\; 6_{{maximum}\; {response}}^{1}} -} \\ {\% \mspace{14mu} {pStat}\; 6_{baseline}^{1}} \end{matrix}} \times 100}$

Dose-response curves were plotted as Y: % of maximum response versus X: concentrations (nM) of huTBTI3_2_1 by SPEED v2.0-LTS to calculate the concentration giving 50% of maximum response (IC₅₀).

Dose response curve was modeled by the four-parameter logistic model:

${\% \mspace{14mu} {Maximum\_ response}} = {c + \frac{d - c}{1 + {\exp \left\{ {b\left( {{\log ({dose})} - {\log (e)}} \right)} \right\}}}}$

The parameters c and d are the lower and upper limits, negative b is the relative slope around e, and the e parameter is IC₅₀ and is the dose producing a response half-way between the upper limit, d and lower limit, c. The four parameters (b,c,d,e) were estimated by non-linear least squares method. SAS procedure NLIN in SAS system release 8.2 for sun solaris via SPEED v2.0-LTS internal software was used. After obtaining the IC₅₀ estimation from each of the three curves, the geometric mean of the 3 IC₅₀ values were calculated.

huTBTI3_2_1 inhibited IL-4-induced Stat6 phosphorylation in donors 245, 229 and 217 with IC_(50s) of 1.32 nM, 0.73 nM, and 0.78 nM respectively. huTBTI3_2_1 inhibited IL-13-induced Stat6 phosphorylation in donors 245, 229 and 002 with IC_(50s) of 2.65 nM, 3.68 nM, and 1.32 nM respectively.

The geometric mean IC₅₀s of huTBTI3_2_1 in inhibiting IL-13 or IL-4-induced Stat6 phosphorylation from 3 separate experiments were 2.34 nM and 0.91 nM, respectively (Table 3).

TABLE 3 IC₅₀ values of huTBTI3_2_1 in inhibiting Stat6 phosphorylation induced by IL-13 or IL-4 in human blood monocytes. IC₅₀ (nM) IC₅₀ (nM) Blood donor no. Inhibition of IL-13 Inhibition of IL-4 245 2.65 1.32 229 3.68 0.73 217 * 0.78 002 1.32 ** Geometric Mean (95% Cl) 2.34 (0.64 to 8.61) 0.91 (0.41 to 2.04) * For donor 217, only IL-4 was tested ** For donor 002, only IL-13 was tested 95% Cl = 95% confidence interval

Example 3: Effect of huTBTI3_2_1 on IL-4- or IL-13-Induced IL-6 Release and Eotaxin Release from Human IPF Pulmonary Fibroblasts

Human lung fibroblast of idiopathic pulmonary fibrosis (IPF) patient, designation LL97A (AIMy), item number CCL-191, F-12K Medium (Kaighn's Modification of Ham's F-12 Medium) and Fetal Bovine Serum (FBS) were from the American Type Culture Collection (ATCC, Manassas, Va.). Albumin from bovine serum (BSA) was from Sigma-Aldrich (St. Louis, Mo.). Recombinant human IL-13 (rhIL-13) was from PeproTech (Rocky Hill, N.J.); Recombinant human IL-4 (rhIL-4) was from R&D SYSTEMS (Minneapolis, Minn.). DuoSet ELISA Development System for human CCL11/Eotaxin and IL-6 were both from R&D SYSTEMS.

LL97A cells at passage 7 were plated on a 96 well cell culture plate at 20,000 cells per well in F-12K Medium with 15% FBS and incubated at 37° C. 5% CO₂ in a humidified incubator for 24 hours. The medium was then replaced with F-12K Medium with 0.1% BSA and the plate was incubated overnight at 37° C., 5% CO₂ in a humidified incubator for serum starvation. Cells were then treated overnight at 37° C., 5% CO₂ in a humidified incubator with a 3-fold serially diluted, 8 concentration points of huTBTI3_2_1 with a combination of 15 ng/ml (1.2 nM) rhIL-13 plus 5 ng/ml (0.36 nM) rhIL-4 in a total volume of 200 μl per well. Each treatment was in triplicate. 150 μl per well of the cell culture supernatant was then taken and diluted into 300 μl F-12K Medium with 0.1% BSA (3-fold dilution) for eotaxin and IL-6 ELISA.

The ELISAs were carried out according to the instructions of DuoSet ELISA Development System for human CCL11/Eotaxin and IL-6 of R&D SYSTEMS. The ELISA plates were read in a SPECTRA_(MAX)340PC plate reader (Molecular Devices) for optical density (OD) at 450 nm and 540 nm. OD values at 540 nm were subtracted from OD at 450 nm before calculation.

CCL11 (eotaxin) and IL-6 levels in supernatants were derived with 4-parameters standard curve in SOFTmax. Sample average without rhIL-13/rhIL-4 stimulation (basal level) was subtracted from each sample and each sample was then compared with sample average of rhIL-13/rhIL-4 stimulation without huTBTI3_2_1 (set as 100% positive) for % positive control. Error bars represent standard error of mean of triplicate biological samples (cell treatment). huTBTI3_2_1 suppressed IL-4/IL-13-stimulated IL-6 release with an IC50=7.8 nM and suppressed IL-4/IL-13-stimulated eotaxin release with an IC50=3.8 nM (FIG. 2).

Example 4: Effect of huTBTI3_2_1 on IL-4- or IL-13-Induced LOX Expression in IPF Pulmonary Fibroblasts

To assess the effects of huTBTI3_2_1 on IL-4 and IL-13 stimulated expression of profibrotic enzymes, mRNA levels of lysyl oxidase (LOX) was measured in a similar experiment as shown in Example 3. LOX gene expression was determined by Taqman and was normalized to the housekeeping gene GAPDH.

Standard Taqman methods were used. Briefly, cells lysates were prepared with the Cells-to-Ct kit (ABI, Catalog No. AM1729). 20× Human GAPDH TaqMan Endogenous Control Primer/Probe Set: Applied Biosystems, Part Number 4310884E, Probe Dye: VIC-TAMRA. 20× human primer probe sets (probes labeled with FAM dye at 5′ end and nonflourescent quencher at 3′ end) was Human LOX: AOD from Applied Biosystems, Gene Name: lysyl oxidase; Assay ID: Hs00184700_m1. Reverse Transcription (RT) was performed on a PELTIER THERMAL CYCLER with 4 block assembly, Model PTC 225, from MJ RESEARCH. The Taqman instrument was 7900HT Fast Real-Time PCR System, Applied Biosystems, Part number: 4330966; Serial number: 279001674. 20 ul of cell lysate per sample (or water for no template control) was added to 80 ul RT master mix (50 ul 2× RT buffer, 5 ul 20× RT Enzyme mix, 25 ul RNase-free water). RT sequence: reverse transcription at 37 degree C. for 60 minutes, RT inactivation at 95 degree C. for 5 minutes, hold at 4 degree C. forever. For Taqman real-time PCR, PCR cocktail was prepared as follows: 10 ul Taqman gene expression master mix (2×), 1 ul Taqman gene expression assay (20×), 1 ul human GAPDH endogenous control (20×), 3 ul water, add 5 ul DNA. Taqman cycling conditions: UDG incubation hold was 1 rep at 50 degree C. for 2 minutes, enzyme activation was 1 rep at 95 degree C. for 10 minutes, and PCR cycle was 40 reps at 95 degree C. for 15 seconds followed by 60 degree C. for 1 minute.

LOX activity results in crosslinking of extracellular collagen and elastin, resulting in stabilization of the extracellular matrix, and its upregulation has been implicated in experimental pulmonary fibrosis (Rodriguez, C. et al., Drug News Perspect. 21:218-224, 2008).

IL-4 and IL-13 induced LOX gene expression, and this expression was inhibited by huTBTI3_2_1 in a dose-dependent manner, with an IC50 of 3-6 nM (FIG. 4). This effect has been observed in pulmonary fibroblasts from at least 3 IPF patients. IL-4 and IL-13 failed to induce genes for fibronectin and insulin-growth factors (IGF; data not shown). This data demonstrate pulmonary fibroblasts from IPF subjects express functional IL-4/IL-13 receptors whose activation by IL-4 and IL-13 results in direct and indirect profibrotic effects. The activation of the profibrotic effects of pulmonary fibroblasts from IPF patients with the cytokines IL-4 and IL-13 is inhibited by huTBTI3_2_1.

Example 5: Effect of huTBTI3_2_1 Against Allergen-Induced Acute Asthma in Cynomolgus Monkeys

Since huTBTI3_2_1 does not bind to rodent IL-4 or IL-13, we were not able to test the molecule for protective effects in rodent models of lung fibrosis. Although huTBTI3_2_1 does bind to cynomolgus monkey IL-4 and IL-13, there are no models of lung fibrosis available in this species. Therefore, to test the ability of huTBTI3_2_1 to inhibit effects of IL-4 and IL-13 in the pulmonary compartment, we investigated its protective effects in a model of acute asthma in a non-human primate species (cynomolgus monkeys). The study used male cynomolgus monkeys (Macaca fascicularis) that are naturally sensitized to Ascaris suum allergens.

To induce airway hyperresponsiveness and airways inflammation, monkeys were challenged with inhaled Ascaris suum extract. Six days before antigen challenge, monkeys received huTBTI3_2_1 (2.5 mg/kg IV), or the same dose of a comparator antibody (anti-IL-13, IMA638) or a control antibody (control antibody does not bind IL-4 or IL-13).

Bronchoconstrictor responses (increases in lung resistance) to ascending doses of inhaled methacholine were measured using a MI² respiratory analyzer. Measurements were made at least 24 hours before challenge, and again 24 hours after challenge. Airway responsiveness was calculated as the provocation concentration of methacholine required to cause a 100% increase in lung resistance (PC₁₀₀). Immediately after measurement of airway responsiveness, bronchoalveolar lavage was performed to allow counts of total leukocytes and eosinophils in the airways. Cell counts were expressed as cell numbers per ml of lavage fluid. The differences ( ) in methacholine PC₁₀₀ values and airway cell numbers before and after antigen challenge were calculated.

Blood samples were collected 24 hours before antigen challenge, and again 7 days after challenge, to allow assay of total immunoglobulin E (IgE) titers. The percent change in IgE titer was calculated.

Antigen challenge caused airway hyperresponsiveness (i.e. a decreased methacholine PC100 (FIG. 4) and an accumulation of total leukocytes and eosinophils in the airways (FIGS. 5 and 6). When compared with control antibody, prophylactic treatment with either huTBTI3_2_1 (2.5 mg/kg) or IMA638 (2.5 mg/kg) significantly suppressed the development of airway hyperresponsiveness. IMA638, but not huTBTI3_2_1, significantly reduced the antigen-induced accumulation of total leukocytes in the airways (FIGS. 5 and 6). huTBTI3_2_1 or IMA638 had no significant effects on the accumulation of eosinophils. huTBTI3_2_1, but not IMA638, significantly reduced the IgE titer (FIG. 7).

Example 6: Effects of huTBTI3_2_1 Against TF-1 Cell Proliferation Induced by Recombinant Human and Cynomologus Monkey IL-4 and IL-13 In Vitro

As a further study of the ability of huTBTI3_2_1 to inhibit IL-4 and IL-13-induced cell activation, the IC₅₀ values for inhibition of TF-1 cell (a human erythrocyte line) proliferation induced by recombinant human IL-4 (hIL-4) and IL-13 (hIL-13) was determined. IL-4- or IL-13-induced TF-1 cell proliferation is commonly used in the literature as an assay for the bioactivities of these cytokines.

TF-1 cells were incubated for 72 hours with huTBTI3_2_1 at a range of concentrations together with either hIL-4 (5 ng/ml), hIL-13 (15 ng/ml), cIL-4 (5 ng/ml) or cIL-13 (30 ng/ml). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added for the final 3 hours as a marker of cell proliferation. Optical density values at 490 nm were then recorded.

huTBTI3_2_1 markedly inhibited hIL-4, cIL-4, hIL-13 and cIL-13-induced TF-1 cell proliferation in a concentration-dependent manner and with comparable potencies. The geometric mean IC₅₀ values were 2.03 nM and 0.53 nM respectively against hIL-4 and cIL-4-induced proliferation, and 3.02 nM and 0.45 nM respectively against hIL-13 and cIL-13-induced proliferation are shown in Table 4.

TABLE 4 Inhibitory effects of huTBTI3_2_1 against TF-1 cell proliferation induced by recombinant human and cynomolgus monkey IL-4 and IL-13 Mean IC₅₀, nM Inhibition of human IL-4 2.03 Inhibition of human IL-13 3.02 Inhibition of cynomolgus 0.53 IL-4 Inhibition of cynomolgus 0.45 IL-13 IC₅₀ = Concentration of SAR156597 that inhibits proliferation by 50%. n = 3 (experimental triplicates).

The results of this study demonstrate that huTBTI3_2_1 neutralizes the biological activities of IL-4 and IL-13 as shown by the decreased cell proliferation of TF-1 cells following stimulation by these cytokines. Hence, targeting these cytokines with huTBTI3_2_1 offers a therapeutic approach that may interrupt the fibrotic process in patients with IPF.

Example 7: Effects of SAR156597 on IL-4 and IL-13 Mediated TGFβ Release

IL-4 and IL-13 have been shown to stimulate TGFβ release from human pulmonary epithelial cells. We determined the effect of SAR156597 on release of this profibrotic cytokine from human small airway epithelial cells (SAEC) and human bronchial epithelial cells (NHBE). SAEC were plated on 12 well plates at 50,000 cells per well in small airway epithelial culture medium (Lonza) and cultured for 3 days. NHBE cells were cultured at 75,000 cells per well in 12 wells plates in BMEM (Lonza) for 3 days. Cells were starved with basal medium containing 5 μg/ml insulin and 5 μg/ml transferrin overnight and then treated with a combination of 15 ng/ml (1.2 nM) rhIL-13 plus 5 ng/ml (0.36 nM) rhIL-4 in the presence of a range of concentrations of SAR156597. TGFβ2 in cell supernatants was determined by ELISA (E-biosciences, cat #BMS254). SAR156597 inhibited IL-4 and IL-3 stimulated TGFβ2 release from NHBE and SAEC cells in a dose-dependent manner (FIG. 8).

Example 8: Pharmacokinetic Study after Repeated Intravenous 5 Minutes Infusions of 2.5 mg/kg of Humanized Bispecific Anti-IL-4/IL-13 (huTBTI3_2_1) Monoclonal Antibody to Cynomolgus Monkeys

In this study the pharmacokinetic properties of huTBTI3_2_1, a humanized bispecific monoclonal antibody (BsAb) to IL-4/IL-13, was measured after repeat dose administration. The goal was to demonstrate that there was an accumulation of huTBTI3_2_1 over time and to monitor the animal production of anti-drug antibodies.

Male Macaca fascicularis (6-8 kg) were obtained from Charles River, Houston, Tex. Route of administration was by i/v perfusion in 5 minutes. Five doses were given serially. Blood samples (1 ml) were taken at the following time points: (first dose) 0 h, 0.5 h, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 240 h; (second, third and fourth dose) 0 h, 0.5 h, 2 h, 24 h; (fifth dose) 0 h, 0.5 h, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 240 h, 336 h, 504 h, 672 h, 840 h, 1008 h (h=hour).

The serum samples were stored at −20° C. until analysis. Two separate assays using enhanced electro-chemiluminescence (EECL) assays with Meso Scale Discovery (MSD) technology were used to determine huTBTI3_2_1 levels in serum (FIG. 9). A third assay was developed to evaluate the anti-drug-antibody (ADA), i.e. anti-bispecific monkey antibodies, response.

The first assay (diagramed in panel A of FIG. 9) was designed to detect the total amount of human antibody in the serum by measuring the concentration of human light chain kappa). In this assay, MSD high binding plates were coated overnight with 1 μg/mL of mouse monoclonal anti-human kappa chain (clone 4G7; Abcam; #ab1936) diluted in PBS. Following an overnight incubation at 4° C. with serum samples diluted 1:1000 and 1:5000, Sulfo-Tag labeled goat anti-human antibody (MSD; #R32AJ-1) at a concentration of 1 μg/mL was added and detected using a MSD plate Sector Imager 6000.

The second assay (diagramed in panel B of FIG. 9) was designed to measure the proportion of bispecific antibody in the serum capable of binding IL-4 and IL-13 (measurement of the amount of functional antibody). In this assay, MSD high binding plates were incubated overnight at 4° C. sequentially with 2 μg/mL of a mouse anti-human IL-4 (clone 4D9; Ancell; #ANC-396), then with 200 ng/mL of recombinant human IL-4 (eBioscience, #34-8049) and finally with monkey sera diluted 1:1000 and 1:5000. Bound bispecific antibodies were revealed by sequential incubation with 200 ng/mL of recombinant human IL-13 (eBioscience; #34-8139, then with 200 ng/mL of biotinylated rabbit polyclonal anti-human IL-13 antibody (eBioscience; #13-7138) and finally with Sulfo-Tag streptavidin (MSD; #R32AA-1) at a concentration of 1 μg/mL.

Thirdly, ADAs were detected with a bridging assay (diagramed in panel C of FIG. 8). In brief, sera diluted 1:10 were incubated overnight at 4° C. with a mixture of biotinylated and Sulfo-tagged huTBTI3_2_1 (2 μg/mL of each final). Complexes were then trapped in streptavidin coated plates (MSD; #L11SA-1) by incubation for 4 hours at room temperature and revealed using a MSD plate Sector Imager 6000.

Standard sample concentrations were prepared in PBS containing 0.5% BSA as indicated in the following table. For the assay to detect the total amount of huTBTI3_2_1 there was no significant difference whether calibration samples were prepared in PBS containing 0.5% BSA, 0.1% monkey plasma or PBS containing 0.5% BSA only. For the assay to detect the fraction of huTBTI3_2_1 specific to IL-4 and IL-13 there was no significant difference whether calibration samples were prepared in PBS containing 0.5% BSA, 1% monkey plasma or PBS containing 0.5% BSA only. Both calibration curves were weighed by 1/x² using a linear regression and shown to be linear within all calibration points (R²>0.98).

TABLE 5 Standard Sample Concentrations Sample Concentrations of Standards Assay Compound ID Matrix (ng/ml) Total BsAb huTBTI3_2_1 PBS, 0.5% 50; 16.7; 5.6; Batch BSA 1.9; 0.6; 0.2; 0.07 #LP08059 BsAb specific huTBTI3_2_1 PBS 0.5% 200; 50; 12.5; 3.1; to IL-4 and IL- Batch BSA 0.8; 0.2; 0.05 13 #LP08059

In parallel to ADA measurements from sera, a data validation curve was obtained by mixing dilutions of a mock ADA with biotinylated and Sulfo-tagged huTBTI3_2_1. That antibody was a mouse anti-human IgG4 (Abcam; #ab1950-1) and showed the best signal to noise ratio out of several antibodies tested.

TABLE 6 Standard Sample Concentrations for ADA Concentrations of Standards Assay Compound ID Sample Matrix (ng/ml) ADA Mock ADA PBS, 0.5% 10000; 2500; 625; 156; 39; 9.7 BSA

The lower limits of quantitation (LLOQ) of huTBTI3_2_1 were 70 ng/mL and 5 ng/mL for total assay and specific assay respectively. The determination of ADA response is not quantitative but qualitative in comparison to the mock ADA response.

The pharmacokinetic parameters were calculated from the arithmetic mean of the serum concentrations/the individual animals following the 5^(th) dose using the program WinNonLin 5.2., non-compartment model 202.

TABLE 7 Total concentrations of bispecific antibodies (BsAb) in monkey sera following repeated intravenous 5 minutes infusions of 2.5 mg/kg of huTBTI3_2_1 (batch #LP08059) in PBS. For each time point, values are mean concentrations out of three independent measurements done in triplicates. Theoretical Theoretical sampling sampling Concentrations of total huTBTI3_2_1 time (h) time (h) [ng/mL] from 1st from last Monkey Monkey Dosing dose dose #1 SD #2 SD mean dose 1 0 0 <LLOQ n.c. <LLOQ n.c. n.c. 0.5 0.5 77000 23000 87100 25000  82100 2 2 60000 13300 109000 45900  84500 4 4 74000 20800 81700 29400  77900 8 8 54800 11900 64100 12000  59500 24 24 60300 15200 77800 41400  69100 48 48 58700 23800 51700 19400  55200 72 72 41400 10600 38600 7290  40000 96 96 45000 2080 48100 19600  46600 120 120 50700 25600 45800 25800  48300 144 144 53500 32400 53900 39800  53700 168 168 31700 12000 34700 19300  33200 240 240 28200 5170 20800 2270  24500 dose 2 336 0 29700 7420 30500 15500  30100 336.5 0.5 114000 27800 125000 9280 119500 338 2 120000 20400 121000 12900 120500 360 24 91600 19900 84000 9590  87800 dose 3 504 0 83700 16400 47200 287  65500 504.5 0.5 165000 18700 120000 18800 143000 506 2 206000 13100 173000 15600 189500 528 24 156000 18700 104000 10100 130000 dose 4 672 0 101000 9300 69400 11300  85200 672.5 0.5 151000 17600 169000 13000 160000 674 2 186000 29200 202000 3990 194000 696 24 164000 49500 135000 8050 150000 dose 5 840 0 134000 11900 91600 361 113000 840.5 0.5 212000 8650 206000 6180 209000 842 2 219000 13100 174000 9480 197000 844 4 183000 2480 143000 11200 163000 848 8 161000 3310 170000 22000 166000 864 24 218000 8340 205000 3340 212000 888 48 141000 5090 114000 5350 128000 912 72 196000 19100 154000 5540 175000 936 96 243000 14800 128000 19300 186000 960 120 97200 14900 159000 5310 128000 984 144 127000 10000 92600 15100 110000 1,008 168 150000 2040 102000 4600 126000 1,080 240 132000 12200 94700 28300 114000 1176 336 101000 19000 67900 13500  84500 1344 504 100000 21300 55400 67500  77700 1512 672 69400 12200 44500 9970  57000 1680 840 61400 19100 31600 4500  46500 1848 1008 51900 14100 22800 4320  37400 n.c. not calculated; LLOQ = 70 ng/mL

TABLE 8 Concentrations of bispecific antibodies (BsAb) reactive to IL-4 and IL- 13 in monkey sera following repeated intravenous 5 minutes infusions of 2.5 mg/kg of huTBTI3_2_1 (batch #LP08059) in PBS. For each time point, values are mean concentrations out of three independent measurements done in triplicates. Theoretical Theoretical Concentrations of sampling sampling functional huTBTI3_2_1 time (h) time (h) [ng/mL] from 1st from last Monkey Monkey Dosing dose dose #1 SD #2 SD mean dose 1 0 0 <LLOQ n.c. <LLOQ n.c. n.c. 0.5 0.5 104000 3370 115000 3510 110000 2 2 80600 1030 152000 5300 116000 4 4 100000 2610 110000 4830 105000 8 8 70400 1200 85800 3930 78100 24 24 74100 1570 116000 4020 95100 48 48 75400 675 73900 126 74700 72 72 52800 57 48600 776 50700 96 96 48900 1300 59500 2120 54200 120 120 71600 2180 63800 802 67700 144 144 76800 2510 77700 725 77300 168 168 45700 883 44800 9690 45300 240 240 34400 183 25700 892 30100 dose 2 336 0 34500 3700 42000 11000 38300 336.5 0.5 143000 7860 175000 26500 159000 338 2 143000 4610 172000 31200 158000 360 24 115000 14900 112000 19500 114000 dose 3 504 0 84600 26400 55500 10100 70100 504.5 0.5 176000 7540 158000 67600 167000 506 2 205000 24300 208000 66500 207000 528 24 145000 18700 112000 20700 129000 dose 4 672 0 93900 9830 69700 14200 81800 672.5 0.5 151000 645 17900 44900 84500 674 2 187000 3340 219000 46500 203000 696 24 161000 9680 156000 6660 159000 dose 5 840 0 118000 5840 94200 3860 106100 840.5 0.5 196000 8620 218000 25300 207000 842 2 211000 6900 195000 23200 203000 844 4 184000 6100 161000 17100 173000 848 8 159000 12000 182000 13200 171000 864 24 218000 31900 194000 18800 206000 888 48 128000 9180 118000 13900 123000 912 72 149000 1910 156000 n.c. 153000 936 96 157000 53400 145000 n.c. 151000 960 120 93300 6350 172000 27300 133000 984 144 117000 1660 98600 1570 108000 1,008 168 137000 6860 116000 20300 127000 1,080 240 114000 1410 98000 11800 106000 1176 336 85100 11400 64400 13300 74800 1344 504 77700 12300 48800 10100 63300 1512 672 47600 978 37100 8250 42400 1680 840 36400 1600 22800 3130 29600 1848 1008 21900 9550 15400 3460 18700 n.c. not calculated; LLOQ = 5 ng/mL

TABLE 9 Pharmacokinetic parameters of huTBTI3_2_1 after repeated intravenous 5 minutes infusions of 2.5 mg/kg of huTBTI3_2_1 (Batch #LP08059) in PBS in monkeys. Parameters were obtained after analysis of the functional BsAb concentration values with WinNonLin 5.2, non-compartment model 202. Intravenous administration (2.5 mg/kg)-1st dose C_(max) AUC_((0-336h)) t_(last) Subject (ng/mL) (ng · h/mL) (h) Monkey #1 104000 17000000 336 Monkey #2 152000 18000000 336 Average 128000 18000000 336 Intravenous administration (2.5 mg/kg)-5th dose C_(max) AUC_((0-1008h)) t_(last) AUC_((0-inf)) AUC_((0-336h)) T_(1/2) Cl Vd_(ss) Subject (ng/mL) (ng · h/mL) (h) (ng · h/mL) (ng · h/mL) (h) (L/h/kg) (L/kg) Monkey #1 220000 79000000 1008 90000000 43000000 360 0.000028 0.014 Monkey #2 220000 65000000 1008 72000000 40000000 290 0.000035 0.014 Average 220000 72000000 1008 81000000 42000000 330 0.000032 0.014 Accumulation ratio (= AUG _((0-336h)) dose 5/AUC _((0-336h)) dose 1) = 2.3

TABLE 10 Anti-drug antibody response to huTBTI3_2_1 after repeated intravenous 5 minutes infusions of 2.5 mg/kg of huTBTI3_2_1 (Batch #LP08059) in PBS in monkeys. Theo- Theo- retical retical sampling sampling MSD signal from serum time (h) time (h) samples 1:10 (arbitrary unit) from 1st from last Monkey Monkey Dosing dose dose #1 SD #2 SD mean dose 1 0 0 769 71 714 71 742 0.5 0.5 565 68 468 50 516 2 2 559 42 547 14 553 4 4 511 39 531 51 521 8 8 477 55 506 45 492 24 24 509 17 497 73 503 48 48 533 93 479 56 506 72 72 689 60 644 55 667 96 96 2000 110 1110 24 1560 120 120 4320 77 1030 103 2680 144 144 5550 677 810 114 3180 168 168 3030 179 663 86 1850 240 240 1720 15 571 25 1150 dose 2 336 0 1270 61 610 15 940 336.5 0.5 925 90 595 20 760 338 2 1000 35 604 78 802 360 24 751 17 716 109 734 dose 3 504 0 965 53 945 27 955 504.5 0.5 668 90 716 5 692 506 2 597 63 585 52 591 528 24 500 48 562 54 531 dose 4 672 0 646 52 646 26 646 672.5 0.5 493 33 593 67 543 674 2 573 60 631 28 602 696 24 644 49 508 70 576 dose 5 840 0 672 48 452 23 562 840.5 0.5 563 39 449 45 506 842 2 530 22 463 20 497 844 4 502 61 449 56 476 848 8 525 61 388 16 456 864 24 505 59 440 46 472 888 48 706 126 564 54 635 912 72 616 59 396 50 506 936 96 618 71 467 44 542 960 120 743 113 393 71 568 984 144 616 68 402 56 509 1008 168 467 28 402 48 435 1080 240 487 9 430 26 459 1176 336 503 25 421 12 462 1344 504 570 69 403 39 487 1512 672 667 47 412 10 540 1680 840 583 33 431 21 507 1848 1008 541 109 400 53 470

The serum exposure of the bispecific antibody huTBTI3_2_1 to IL-13/IL-4 was measured in male cynomolgus monkeys to establish repeat dose pharmacokinetic parameters.

huTBTI3_2_1, at a dose of 2.5 mg/kg, was given repeatedly to cynomolgus monkeys via intravenous infusions in a serial sampling paradigm. The antibody shows accumulation from the 1^(st) to the 5^(th) dose. C_(max) and partial AUC_(0-336h) increase from 128,000 ng/mL to 220,000 ng/mL and 18,000,000 ng·h/mL to 42,000,000 ng·h/mL respectively. After the 5^(th) dose there is good exposure to the antibody with AUC_(0-inf) of 81,000,000 ng·h/mL (see FIG. 10). The antibody shows a serum clearance of 0.000032 L/hr-kg and a volume of distribution of 0.014 L/kg. The terminal elimination half-life is found to be 330 hours.

One monkey of the study shows a transient anti-drug antibody (ADA) response, which peaks on days 5-7. ADA level is back to background level by the day of the second infusion. There is no significant rise of ADA levels thereafter. The other monkey displays no significant levels of ADA at anytime.

No particular clinical sign or weight loss was observed during or after administration.

Example 9: Study Design Summary for a Randomized, Double-Blind, Placebo-Controlled Study of the Safety, Tolerability, and Pharmacokinetics of Ascending Single Subcutaneous Doses of huTBTI3_2_1 in Healthy Young Male Subjects

This is the first investigation of huTBTI3_2_1 in humans and involves careful dose escalation in healthy subjects to obtain initial information on the safety, tolerability, and pharmacokinetic (PK) of single subcutaneous (SC) doses. Dose escalation was conducted in cohorts of healthy young male subjects (18 to 45 years of age, body weight between 50.0 and 95 kg, body mass index between 18.0 and 30.0 kg/m²; certified as healthy by a comprehensive clinical assessment; normal heart rate and blood pressure after 10 minutes resting in supine position: 95 mmHg <systolic blood pressure <140 mmHg; 45 mmHg <diastolic blood pressure <90 mmHg; 40 bpm <heart rate <100 bpm; Normal standard 12-lead ECG after 10 minutes resting in supine position; 120 ms<PR<220 ms; QRS <120 ms; QTc≤430 ms; laboratory parameters within normal range; C-reactive protein should not exceed 3 mg/L (using a high-sensitivity method of measurement); cardiac troponin I must not exceed the upper laboratory norm.)

This was a single-center, randomized, double-blind, placebo-controlled, ascending single SC dose study in four sequential cohorts of healthy young male subjects. Each dose cohort/group was designed to consist of 8 subjects (6 receiving huTBTI3_2_1 and 2 receiving placebo). This stepwise, dose escalation design was typical for introduction of a new therapeutic entity into humans.

The observation period of 85 days (˜12 weeks) after dosing for treatment-emergent adverse events (TEAEs) and PK analysis was appropriate taking into account a typical elimination half-life for a monoclonal antibody of about 15 days. If ADA or autoimmunity antibodies (rheumatoid factor [RF], antinuclear antibodies [ANA], or anti-neutrophil cytoplasmic antibodies [ANCA]) measured at 12 weeks after dosing were increased from baseline, then an additional follow-up visit would occur at 6 months after dosing to document resolution or longer-term persistence. If, at the end of dose escalation in four sequential dose cohorts, additional information was needed at doses below the highest administered dose, then a fifth dose cohort of 8 subjects will be dosed and evaluated.

Given the immunomodulatory mechanism of action of huTBTI3_2_1, several specific laboratory tests related to inflammation were implemented.

C-Reactive Protein (CRP):

Inflammation is associated with elevations in acute phase proteins such as CRP. Using high sensitivity methodology, the upper limit of normal (ULN) for high sensitivity C-reactive protein (hsCRP) is now often stated to be 3 mg/L for the purpose of assessing cardiovascular risk; however, about a third of healthy subjects in the United States have CRP values between 3 and 10 mg/L, with occasional spurious elevations between 10 and 15 mg/L (Kushner I., et al. Am J Med 2006; 119(2):166.e17-28; Ridker, P M, et al., Circulation 2003:107(3):391-7; Pearson T A, et al, Circulation 2003:107(3):499-511; Ridker P M et al, 2000:342(12):836-43; Unek, I T et al., Clin Med Res. 2010; 8(2)89-95). Using serial sampling from healthy subjects, the critical difference for sequential values significant at P<0.05 (ie, the smallest percentage change unlikely to be due to analytical variability or normal within-subject variability) has been reported to be 118% (Macy E M et al, Clin Chem 1997:43(1):52-8). C-reactive protein values are typically considered clinically significant at levels above 10 mg/L, and sustained elevations above this level may be cause for concern. During acute inflammatory responses, values exceeding 100 mg/L can be observed (Clin B and Olshaker J S, J. Emerg Med. 1999: 17(6)1019-25). Elevation in CRP has been consistently reported for conditions of active vasculitis (Konttinen Y T et al, Ind J Rheumatol 2007:2(3):100-4Hesselink D A et al., Scand J Rheumatol 2003:32(3)151-5). For this protocol, hsCRP was used, along with clinical observations, as the primary instrument for detection of drug-related inflammation and possible vasculitis. A sustained elevation in hsCRP above 10 mg/L for at least 72 hours, was viewed as possible early evidence of vasculitis. In general, drug-induced vasculitis is reversible upon discontinuation of treatment, and markers of inflammation such as hsCRP should return to normal or baseline values (Wiik, A, Curr Opin Rheumatol 2008:20(1)35-9; Calabrese L H and Duna G F, Curr Opin Rheumatol 1996; 8(1)34-40). In addition, consecutive elevations above 20 mg/L after drug administration can be viewed as evidence of a possible drug-related pro-inflammatory stimulus and, after excluding other causes of inflammation such as acute infections, possible reason for cessation of further dosing.

Cardiac Troponin I:

Serum cardiac troponin I (cTnI) was monitored to detect myocardial injury caused by potential coronary vasculitis (Kim M and Kim K, Pediatr Cardiol, 1999:20(3): 184-8). Interpretation of cTnI in the study was made in the context of clinical signs, symptoms, ECG and hsCRP, as cTnI elevations may be caused by factors other than myocardial injury (including strenuous exercise) (Wu A H, et al. Clin Chem. 2007; 53(12):2086-96).

Supplementary Tests for Vasculitis:

To further characterize inflammation associated with sustained elevations in hsCRP in individual subjects, additional laboratory tests related to vasculitis were performed, including tests for complement (C3, C4, and CH₅₀), cryoglobulin, ANCA (immunofluorescence for perinuclear and cytoplasmic ANCA and confirmatory immunoassays for anti-protease 3 [PR3] and anti-myeloperoxidase), RF, and ANA. Baseline values were established prior to dosing for all subjects; further assay of these supplementary tests for vasculitis were done for individual subjects who showed sustained elevation in hsCRP (>10 mg/L for at least 72 hours), in which case the supplementary tests were performed immediately (as soon as practicable) and at several subsequent visits.

The proposed array of supplementary tests provided insight into the mechanism of vasculitis, should it occur. ANCA is a new classification criteria (Sunderktter C and Sindrilaru A. Eur J Dermatol. 2006; 16(2):114-24; Watts R et al., Ann Rheum Dis. 2007; 66(2):222-7; Watts R A et al., Rheumatology (Oxford). 2010 Jul. 20: 1-3). ANCA are classified according to the indirect immunofluorescence (IIF) patterns they produce on normal neutrophils and according to their target antigens (Pollock W et al. J Immunol Methods. 2009; 347(1-2):19-23; De Rosa F G and Agnello V. J Rheumatol. 2009; 36(9):1953-5; Clin Sci (Lond). 2005; 108(2):101-12) If myeloperoxidase-ANCA is positive, Churg-Strauss syndrome or microscopic polyarteritis can be suspected. If PR3-ANCA is positive Wegener's granulomatosis is most likely. If ANCA test is negative and cryoglobulin test is positive, cryoglobulinemic vasculitis should be suspected and its underlying diseases should be ruled out, particularly hepatitis C and B, systemic lupus erythematous (SLE), and Sjogren's syndrome. Serum C3 and C4 are often consumed in cryoglobulinemia, but are usually normal in polyarteritis nodosa as well as ANCA vasculitis. ANCA can be positive in the presence of other diseases including infection, inflammatory bowel disease and other connective tissue disease (eg, rheumatoid arthritis). In these cases, ANCA are positive but are negative for PR3 and myeloperoxidase.

Vascular Endothelial Activation Biomarkers:

The development of vasculitic lesions is associated with activation of endothelial cells and neutrophils (Tesfamariam B and DeFelice A F. Vascul Pharmacol. 2007; 46(4):229-37; Toxicol Appl Pharmacol. 2005; 207(2 Suppl):441-5). Enhanced expression of adhesion molecules such as e-selectin promotes interaction of the endothelium with circulating inflammatory cells. Various endothelial activation markers such as endothelin-1 and thrombomodulin are reportedly elevated during vasculitis. Vascular endothelial growth factor (VEGF) can alter vascular permeability and is elevated in serum from patients with Behçet's disease, microscopic polyangiitis, polyarteritis nodosa, giant cell arteritis, and systemic vasculitis (Cekmen M et al., Int J Dermatol. 2003; 42(11):870-5). If treatment-emergent inflammation (eg, sustained hsCRP elevations) or changes in laboratory values consistent with vasculitis was noted in multiple subjects, then archival serum and plasma samples (collected before and periodically after drug administration) was assayed for various exploratory biomarkers associated with vascular endothelial activation to further characterize the nature of the inflammation.

Lymphocyte Subsets:

To assure that specific subtypes of human lymphocytes are not selectively affected by huTBTI3_2_1, lymphocyte subsets were assessed using flow cytometry. This will include total T cells, T helper cells (CD4), T suppressor cells (CD8), and total B cells (CD19) expressed as absolute numbers and as a percent of total lymphocytes, as well as the CD4/CD8 ratio.

Immunogenicity

Systemic administration of monoclonal antibodies is associated with generation of ADA which can alter the PK and/or activity of the therapeutic antibodies (Hansel T T et al. Nat Rev Drug Discov. 2010; 9(4):325-38). Immunogenicity was assessed using an enzyme-linked immunosorbent assay (ELISA) for anti-huTBTI3_2_1 antibodies; a functional assay for assessment of antibody neutralization of huTBTI3_2_1 may be employed during future studies.

Urinary Albumin:

Appearance of protein in the urine is an indication of increased permeability of the renal glomeruli and, during clinical drug trials, evidence of possible renal injury. A standard urine dipstick assay for protein is typically used for this purpose. However, dipstick methodology may not detect the more subtle changes in glomerular function as might occur during early stages of vasculitis. Therefore, a more sensitive assay for urinary albumin was used during initial clinical studies of huTBTI3_2_1. An early morning spot urine collection was used to monitor potential appearance of microalbuminuria and reported as the albumin/creatinine ratio to correct for fluctuations in the extent of urine solute dilution. Post-treatment appearance of microalbuminuria was defined as observation of an albumin/creatinine ratio >30 μg/mg in 2 of 3 consecutive urine collections, as recommended for the monitoring of patients with diabetes mellitus (American Diabetes Association. Standards of medical care in diabetes—2009. Diabetes Care. 2009; 32 Suppl 1:S13-61). Since exercise can transiently elevate urinary albumin, vigorous physical exercise must be restricted prior to urine collection (Heathcote K L et al., Clin Invest Med. 2009; 32(4):E261-5). Urinary albumin results obtained within 24 hours of vigorous physical exercise must be excluded from consideration for purposes of defining microalbuminuria. An observation of an albumin/creatinine ratio >300 μg/mg in 2 of 3 consecutive urine collections was assessed as evidence of macroalbuminuria (American Diabetes Association. Standards of medical care in diabetes—2009. Diabetes Care. 2009; 32 Suppl 1:S13-61).

Tolerability at the Site of Investigational Product (IP) Injection:

The degree of discomfort and tissue reaction at the site of IP injection was monitored for up to 2 weeks after dosing, including standard qualitative and quantitative assessments for present pain (verbal scale) (Melzack R. The McGill Pain Questionnaire: major properties and scoring methods. Pain 1975; 1:277-299) and for erythema and swelling/induration/edema (Guidance for industry: toxicity grading scale for healthy adult and adolescent volunteers enrolled in preventive vaccine clinical trials, US Dept of Health and Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research, September 2007).

The dose escalation steps for TDU11325 are provided in Table 11. The dose escalation ratio is 2-fold at each escalation step. This serial increase in dose is typical for initial clinical trials of therapeutic monoclonal antibodies and is supported by careful monitoring of potential safety signals.

TABLE 11 Sequential subcutaneous doses of huTBTI3_2_1 Total volume Dose in mg/kg injected assuming body Incremental Dose cohorts subcutaneously weight of 60 kg increase 10 mg 0.1 mL 0.17 mg/kg 20 mg 0.2 mL 0.33 mg/kg 2 40 mg 0.4 mL 0.67 mg/kg 2 80 mg 0.8 mL 1.33 mg/kg 2 150 mg  1.5 ml  2.50 mg/kg 1.9 300 mg  3.0 ml   5.0 mg/kg 2

Higher doses of huTBTI3_2_1 could be tested depending on the results of the lower doses. Higher doses could include any dose lower than, equal to or higher than a 300 mg cohort. Additional dose cohorts contemplated include but are not limited to 175 mg, 200 mg, 225 mg, 250 mg, 275 mg and 300 mg. Additional dose cohorts could also be 300 mg, 350 mg, 400 mg or higher.

huTBTI3_2_1 or placebo was administered as periumbilical SC injections in a fasted condition in a zone 4 to 10 cm to the right or left of the umbilicus and above the waistline. The dose cohorts in TDU11325 were obligatorily initiated as smaller subgroups as a safety precaution. For each dose cohort, 2 subjects were dosed on the first day. The remaining subjects in the cohort were dosed no sooner than 2 days (˜48 hours) later, with no more than 2 subjects dosed each day.

A decision to proceed from dose “n” to the next higher “n+1” dose was made jointly by the Sponsor and the Investigator based on a preliminary safety report provided by the Investigator which includes blinded safety data for at least 21 days postdose (Day 22) of at least 6 out of 8 subjects of dose level cohort “n”. Thus, taking into account the staggered dosing within a cohort, a new dose cohort was initiated about every 4 weeks. The relevant data for this decision should be at least: adverse events, hematology, lymphocyte subsets, coagulation, urinalysis, serum biochemistry (including hsCRP and cTnI), ECG, blood pressure, heart rate, and body temperature. The available PK data was also reviewed during the study progression.

In addition to the classic assessment of serious adverse events and the occurrence/severity of other adverse events by the Sponsor and the Investigator, after exploring potential confounding factors, the following criteria were considered as guidance for the decision to stop dosing:

-   -   More than 1 adverse event (same verbatim) of severe intensity         for which the relationship to treatment cannot be reasonably         excluded. The definition of “severe” intensity for an adverse         event is that it prevents daily activities and requires         symptomatic treatment;     -   QTc ≥500 ms;     -   hsCRP >20 mg/L sustained for 2 consecutive blood collections ≥24         hours apart         -   Note: If hsCRP is >20 mg/L at a scheduled collection             timepoint, an additional blood sample should be collected 24             hours later (or as soon as possible thereafter) for             retesting.

huTBTI3_2_1 in lyophilized form for preparation of SC dose solution with each vial containing 185 mg of huTBTI3_2_1 plus excipients and stored between 2° C. and 8° C. (36° F. and 46° F.). To be reconstituted on the morning of dosing (no more than 1 hour prior to SC injection) with 1.7 mL sterile, nonpyrogenic distilled water at room temperature. The concentrations of the constituents in solution after reconstitution for injection were: 100 mg/mL of huTBTI3_2_1 in 6.3 mmol/L monobasic sodium phosphate, 3.7 mmol/L tromethamine, 5% (weight/volume) sucrose, 3% (w/V) proline, and 0.2% (w/V) polysorbate 80 with a final pH of 7.0. For placebo, each vial will contain 2 mL of liquid consisting of the same excipients at the same concentrations as for the reconstituted huTBTI3_2_1 formulation.

TABLE 12 Planned huTBTI3_2_1 subcutaneous administration Group Dose (mg) Total volume injected 1 10 0.1 mL 2 20 0.2 mL 3 40 0.4 mL 4 80 0.8 mL 5 150 1.5 mL 6 300 3.0 mL (two 1.5 mL injections)

A predose fasting period commenced at least 10 hours prior to dosing and continued for 2 hours after dosing.

The safety and tolerability investigations at baseline and during the study consisted of:

-   -   Physical examination (includes at least: heart and respiratory         auscultation; peripheral arterial pulse in both arms and both         legs; pupil, knee, Achilles, and plantar reflexes; peripheral         lymph nodes and abdomen examination; inspection of skin, hands         and feet);     -   Body weight (kg);     -   Posteroanterior chest x-ray (at screening only unless clinically         indicated postdose)     -   Body temperature (either oral or tympanic but consistently the         same method throughout the study for all subjects);     -   Heart rate and systolic and diastolic blood pressure measured         after 10 minutes in supine resting position and after 3 minutes         in standing position);     -   Laboratory tests (all blood samples collected in the morning         under fasted conditions, ie, only water for at least 10 hours         prior, unless otherwise specified):         -   Hematology: red blood cell count (RBC), hematocrit (Hct),             hemoglobin (Hb), white blood cell count (WBC) with             differential (neutrophils, eosinophils, basophils, monocytes             and lymphocytes), platelets;         -   Coagulation: prothrombin time (PT), international normalized             ratio (INR), fibrinogen, and activated partial             thromboplastin time (aPTT);         -   Serum biochemistry:             Electrolytes: sodium, potassium, chloride, calcium;             Liver function: AST, ALT, alkaline phosphatase,             gamma-glutamyl transferase (GGT), total and conjugated             bilirubin;             Renal function: urea, creatinine;             Metabolism: glucose, albumin, total protein, total             cholesterol, triglycerides;             Potential muscle toxicity: creatine kinase (CK);             Potential cardiac toxicity: cTnI;             Inflammation biomarker: high-sensitivity CRP (hsCRP);     -   Lymphocyte subsets:         Total T cells (CD3), T helper cells (CD4), T suppressor cells         (CD8), and total B cells (CD19) by flow cytometry, expressed as         absolute numbers and as percent of total lymphocytes, as well as         the CD4/CD8 ratio.     -   Immunogenicity: anti-huTBTI3_2_1 antibodies;     -   Supplementary tests for vasculitis (at baseline and, then         postdose only for subjects exhibiting hsCRP >10 mg/L for >72         hours):         Complement assays: C3, C4, CH₅₀;

Cryoglobulin;

Rheumatoid factor (RF); Anti-nuclear autoantibodies (ANA): HEp2 IIF (titer and pattern); Anti-neutrophil cytoplasmic autoantibodies (ANCA): immunofluorescence for perinuclear and cytoplasmic ANCA and confirmatory immunoassays for anti-protease 3 and anti-myeloperoxidase.

-   -   Serology tests: HBsAg, hepatitis B core antibody, anti-HCV         antibodies, anti-HIV1 and anti-HIV2 antibodies;     -   Archival blood samples: Serum and plasma samples were prepared         and stored for future assay of laboratory parameters needed to         support safety assessment. In particular, an observation of         sustained inflammation (eg, hsCRP >10 mg/L for at least 72         hours) or appearance of treatment-emergent markers of vasculitis         in multiple subjects may lead to assay of exploratory biomarkers         of vascular endothelial activation.     -   For serum, a 10 mL blood sample was collected into a dry, red         topped tube and, within 15 minutes of collection, centrifuged at         approximately 1500 g for 10 minutes at 4° C.; the serum was then         be transferred in roughly equal aliquots (using a plastic         transfer pipet) into 3 storage tubes, which was immediately         capped and frozen in an upright position at −20° C. or colder;     -   For plasma, a 10 mL ethylenediaminetetra-acetic acid (EDTA)         blood sample was collected into a purple-top tube and, within 15         minutes of collection, centrifuged at approximately 1500 g for         10 minutes at 4° C.; the plasma was then be transferred in         roughly equal aliquots (using a plastic transfer pipet) into 3         storage tubes, which was immediately capped and frozen in an         upright position at −20° C. or colder;     -   Urinalysis:         -   A midstream urine specimen was collected and subjected to             the following analyses.         -   A reagent strip dipstick analysis for detection of protein,             glucose, blood (heme), leucocytes, ketone bodies, and pH. A             positive glucose result should be confirmed with an             alternative methodology for glucose according to the local             clinical laboratory standard operating procedures. Upon             initial detection of protein, glucose, or blood in a             subject's urine, an additional urine specimen should be             collected and subjected to urinalysis for confirmation.         -   The urine sediment should be examined for, at a minimum, the             following formed elements: red blood cells, dysmorphic red             blood cells, white blood cells, renal epithelial cells,             casts (hyaline, RBC, WBC, granular, waxy, fatty, renal             cell), bacteria, yeast and trichomonas. Abundance of each of             these elements should be scored using standard laboratory             terminology and units in use at the local laboratory (eg,             number of cells per high power field, few, abundant). Any             additional unusual observations should be noted on reports.         -   Urine microalbumin: quantitative assay of albumin and             creatinine and calculation of albumin/creatinine ratio             (μg/mg).     -   Urine drug screen: amphetamines/methamphetamines, barbiturates,         benzodiazepines, cannabinoids, cocaine, opiates;     -   Alcohol breath or blood test;     -   Adverse events, spontaneously reported by the subject or         observed by the Investigator, were monitored for definition of         adverse events and related responses);     -   Standard 12-lead ECGs.         -   When vital signs, ECG, and blood samples were scheduled at             the same time as an Investigational product administration             and/or a meal, they were done prior to IP intake and/or             meal. Whenever measurements of vital signs, ECG, and blood             sampling for PK or safety coincided, the following order was             respected: ECG, vital signs, PK samples, and safety samples.         -   Twelve-lead ECGs were recorded after at least 10 minutes in             supine position using an electrocardiographic device. The             electrodes were positioned at the same place for each ECG             recording throughout the study (attachment sites of the             leads will be marked with an indelible pen).         -   Each ECG consisted of a 10 second recording of the 12 leads             simultaneously, leading to:             -   One single 12-lead ECG (25 mm/s, 10 mm/mV) print-out                 with heart rate, PR, QRS, QT, QTc automatic correction                 evaluation, including date, time, initials and number of                 the subject, signature of the research physician, and at                 least 3 complexes for each lead. The Investigator                 medical opinion and automatic values were recorded in                 the eCRF. This printout was retained at the site level.                 As an exception, recordings were obtained in triplicate                 on Day 1 pre-dose.

The skin around the periumbilical area was examined for potential reactions to the SC injection. The maximum diameter of erythema and swelling (including induration and/or edema) was measured separately in millimeters and recorded. Erythema and swelling was graded separately as follows in a manner similar to the FDA guidance on assessment of vaccines. If there is no treatment-emergent change in a parameter at the time of observation, a value of 0 was recorded.

TABLE 13 Toxicity grading scale for local tolerability Potentially Life Mild Moderate Severe Threatening Erythema/ 2.5-5.0 cm 5.1-10.0 cm >10.0 cm Necrosis or Redness exfoliative dermatitis Swelling/ 2.5-5.0 cm 5.1-10.0 cm >10.0 cm or Necrosis Induration/ and does not or interferes prevents Edema interfere with with activity daily activity activity

In addition, the degree of itching and appearance of papules, pustules, and vesiculation was each scored using the following grading scale:

-   -   0=None; 1=Hardly perceptible; 2=Mild; 3=Moderate; 4=Severe

Skin reactions of moderate intensity or worse were reported as adverse events. The presence or the absence of the following symptoms or superficial observations were recorded without grading: erosion, dryness, scaling, cracking, scabbing, and glazing.

In addition, the present pain intensity was recorded using the following verbal numeric rating scale (self assessment by the study subject) based on a subset of the McGill Pain Questionnaire. A subject-assessed pain score of ≥3 will be reported as an adverse event.

-   -   0=No Pain; 1=Mild; 2=Discomforting; 3=Distressing; 4=Horrible;         5=Excruciating

Pharmacokinetics

All blood collections for huTBTI3_2_1 PK analysis were scheduled to occur within ±15% of the sampling times. The number of plasma samples by subject and the total number of samples for the study are given in Table 14.

TABLE 14 TDU part - number of plasma samples collected for huTBTI3_2_1 assays Total by subject  17 Total for study (32 subjects)^(a) 544 ^(a)Four (4) dose groups of 8 subjects; not including optional 5th dose group

TABLE 15 Summary of sample handling procedures for huTBTI3_2_1 assays Blood Sample Volume 4 mL Anticoagulant Sodium citrate Plasma Aliquot Split Yes Plasma Storage Conditions ≤−20° C. Plasma Shipment Conditions On dry ice

An ELISA was used for the quantification of huTBTI3_2_1 in human plasma. Biotinylated IL-4 coated on a streptavidin plate is used to capture huTBTI3_2_1, which is then detected by SulfoTag-IL-13. This format, which uses electrochemiluminescence detection, is able to detect huTBTI3_2_1 that retains at least 1 unoccupied binding site for IL-4 and 1 unoccupied binding site for IL-13. Since huTBTI3_2_1 plasma concentrations were typically present in large molar excess compared to concentrations of IL-4 and IL-13, the assay reflected total concentrations of huTBTI3_2_1.

The potential interference of ADA with huTBTI3_2_1 measurements were taken into account when assaying clinical samples.

TABLE 16 Summary of bioanalytical method for huTBTI3_2_1 Analyte huTBTI3_2_1 Matrix Plasma Analytical Technique ELISA Lower limit of Quantification 50 ng/mL (to be confirmed at the end of validation) Analyte huTBTI3_2_1 Assay volume 100 μL Site of Bioanalysis Berlin Pharma (CRO) Saclay, France Method Reference DOH0850 (validation on going) ELISA = enzyme-linked immunosorbent assay

For the analysis of potential anti-huTBTI3_2_1 antibodies (ADA) in human plasma, a bridging qualitative ELISA using electrochemiluminescence detection was used. A cut-off that should provide a 5% false positive rate, above which a plasma sample was considered as potentially positive for anti-huTBTI3_2_1 antibody, was used in each screening assay.

Positive samples in screening assay were then tested in a confirmatory assay (competition with huTBTI3_2_1) in order to demonstrate the presence of antibodies and eliminate false positive results generated from the initial screening assay. Interference of huTBTI3_2_1 in the ADA assay was be documented so that the highest drug concentration that did not affect the limit of ADA detection was known and the interpretation of immunogenicity taken into account this parameter.

TABLE 17 Summary of bioanalytical method for anti-huTBTI3_2_1 antibodies Analyte Anti-huTBTI3_2_1 antibodies Matrix Plasma Analytical Technique ELISA (screening and confirmatory assays) Sensitivity <100 ng/mL Assay volume 100 μL Site of Bioanalysis Montpellier, France Method Reference DOH0851 ELISA = enzyme-linked immunosorbent assay

Plasma concentrations were used to determine the PK parameters of huTBTI3_2_1 listed in Table 18 using standard non-compartmental techniques.

TABLE 18 List of pharmacokinetic parameters for plasma huTBTI3_2_1 and definitions Parameters Definition/Calculation C_(max) Maximum plasma concentration observed t_(max) First time to reach C_(max) AUC_(last) Area under the plasma concentration versus time curve calculated using the trapezoidal method from time zero to the real time AUC Area under the plasma concentration versus time curve extrapolated to infinity according to the following equation: ${AUC} = {{AUC}_{last} + \frac{C_{last}}{\lambda_{Z}}}$ t_(last) Time corresponding to the last concentration above the limit of quantification, C_(last) t_(1/2z) Terminal half-life associated with the terminal slope (λz) determined according to the following equation: $t_{{1/2}Z} = \frac{0.693}{\lambda_{Z}}$ where λz is the slope of the regression line of the terminal phase of the plasma concentration versus time curve, in semi- logarithmic scale. Half-life is calculated by taking the regression of at least 3 points. CL/F Apparent total body clearance of a drug from the plasma calculated using the following equation: ${{CL}\text{/}F} = \frac{Dose}{AUC}$ Vss/F Apparent volume of distribution at steady state after non- intravenous administration Vss/F = CL/F *MRT

TABLE 19 Sampled blood volume Volume per Number of Type sample samples Total Serology tests  5 mL 1  5 mL Hematology  3 mL 13^(a ) 39 mL Serum biochemistry with hsCRP 10 mL 13^(a ) 130 mL  and cTnI Coagulation (PT, INR, fibrinogen,  3 mL 13^(a ) 39 mL and aPTT) Lymphocyte subsets  4 mL 6 24 mL Pharmacogenetic for stored DNA  6 mL 1  6 mL Pharmacokinetic huTBTI3_2_1  4 mL 17  68 mL huTBTI3_2_1 immunogenicity  4 mL 5 20 mL (ADA) Archival sample - serum 10 mL 5 50 mL Archival sample - EDTA plasma 10 mL 5 50 mL Supplementary vasculitis tests 10 mL  6^(b) 60 mL (frozen serum) Cryoglobulin (room temperature 10 mL  6^(b) 60 mL serum) TOTAL 551 mL  ^(a)Includes optional 6-month visit ^(b)Includes 5 additional samples (beyond baseline on Day −1) to be collected only if elevations in hsCRP >10 mg/L for >72 hours are observed. Abbreviations: ADA = anti-drug antibodies; aPTT = activated partial thromboplastin time; cTnI = cardiac troponin I; EDTA = ethylenediaminetetra-acetic acid; hsCRP = high sensitivity C-reactive protein; INR = international normalized ratio; PT = prothrombin time

For C_(max), AUC_(last), AUC, dose proportionality was assessed using the empirical power model (PK parameter=α×dose^(β)), along with an “estimation” interpretation, according to the recommendations of Gough et al Pharmacokinetics UK Joint Working Group Drug Infor J 1995:29:1039-1048.

The power model will be fit on the log-transformed scale:

log(parameter)=log(α)+β×log(dose)+Error.

Model lack-of-fit was assessed by residual plots, and by an F-test of the residual mean square versus the pure error residual mean square. If the model fit is adequate, estimates with 90% confidence intervals for β were obtained, and further used to obtain estimates and 90% confidence intervals for the PK parameter increase associated with an r-fold (r=2 and r=high dose/low dose) increase in dose, by exponentiating r to the powers of the β estimate (“b”) and confidence limits:

r ^(b±t×SE(b))

If there is evidence of model lack-of-fit, then attempts were made to fit the model over a reduced dose range (eg, exclude 1 extreme dose level). Otherwise, a fixed effect model was used, with fixed term for dose, using logarithms of the relevant PK parameters. Estimates with 90% confidence intervals for the parameter increases associated with pairwise dose increases were obtained by first computing estimates with confidence intervals for differences between pairwise dose groups in the fixed effects model framework, and then converting to ratios using the antilog transformation.

For t_(1/2z), dose effect will be assessed with a linear fixed effect model,

Log(t _(1/2z))=Dose+Error

Point estimate and 90% confidence interval for the geometric mean of t_(1/2z) were provided pooled across dose levels and separately for each dose group. The distribution of t_(max) values was represented by histogram plots for each dose level.

Definition of Adverse Event and Serious Adverse Event

An adverse event is any untoward medical occurrence in a subject administered a pharmaceutical product and which does not necessarily have to have a causal relationship with this treatment.

A serious adverse event is any untoward medical occurrence that at any dose:

-   -   Results in death or     -   Is life-threatening, or         -   Note: The term “life-threatening” in the definition of             “serious” refers to an event in which the subject was at             risk of death at the time of the event; it does not refer to             an event which hypothetically might have caused death if it             were more severe.     -   Requires inpatient hospitalization or prolongation of existing         hospitalization, or     -   Results in persistent or significant disability/incapacity, or     -   Is a congenital anomaly/birth defect, or     -   Is a medically important event:         -   Medical and scientific judgment should be exercised in             deciding whether expedited reporting is appropriate in other             situations, such as important medical events that may not be             immediately life-threatening or result in death or             hospitalization but may jeopardize the subject or may             require intervention to prevent 1 of the other outcomes             listed in the definition above.

Note: As guidance for determining which conditions are medically important events, the following examples are provided, though not intended to be exhaustive:

intensive treatment in an emergency room or at home for allergic bronchospasm; blood dyscrasias such as agranulocytosis, aplastic anemia, bone marrow aplasia, myelodysplasia, pancytopenia; convulsions; alanine aminotransferase (ALT) >3×upper limit of normal range (ULN) associated with total bilirubin >2×ULN; asymptomatic ALT increase >10×ULN; or development of drug dependency or drug abuse. Adverse events requiring the Sponsor to be informed immediately:

-   -   ALT increase 2×ULN     -   hsCRP >10 mg/L for ≥72 hours     -   cardiac troponin I (cTnI) >2×ULN     -   If cTnI >2×ULN is observed, cTnI, creatine kinase (CK) and         myocardial B fraction of creatine kinase (CK-MB) values should         be serially tracked (at a minimum in an immediate additional         blood collection and on the following day), along with         additional ECG recordings, until test results return to normal.         Consultation of a cardiologist should be considered promptly if         elevations in cTnI persist, the CK-MB/CK ratio is elevated,         and/or treatment emergent abnormalities in ECG recordings are         observed.     -   QTc ≥500 ms     -   In occurrences of prolongation of QTc automatic measurement ≥500         ms, confirmed by a manual reading by the Investigator, or a         physician delegated by the Investigator, using the Fridericia         formula for correcting QT, the subject should be placed under         supervision in a specialized setting. Appropriate blood samples         will be collected (eg, for cTnI). Subsequent ECG monitoring of         the subject should then be performed on a regular and clinically         responsible basis until the QTc interval returns to a safe value         as determined by the Investigator in agreement with the Sponsor.     -   Severe skin reactions local to the site of IP injection     -   Symptomatic overdose with IP         -   An overdose (accidental or intentional) with the IP is an             event suspected by the Investigator or spontaneously             notified by the subject (not based on systematic IP vial             count) and defined as at least twice of the intended dose.             Laboratory abnormalities include:     -   Neutropenia,         -   Defined as neutrophil blood count <1500/mm³ (but <1000/mm³             in subjects of African descent)     -   Thrombocytopenia,         -   Defined as platelet count <100 000/mm³     -   Acute renal failure         -   Rapid increase in serum creatinine over 150 μmol/L (1.7             mg/dL)         -   Suspicion of rhabdomyolysis

Example 10: Results from the Randomized, Double-Blind, Placebo-Controlled Study of the Safety, Tolerability, and Pharmacokinetics of Ascending Single Subcutaneous Doses of SAR1 56597 in Healthy Young Male Subjects (TDU11325)

The following Tables, Tables 20-35, summarize the data from TDU11325 study.

TABLE 20 Subject disposition SAR156597 10 20 40 80 150 300 Placebo mg mg mg mg mg mg Randomized 12 6 6 6 6 6 6 and treated PGM = PRODOPS/SAR156597/TDU11325/CSR/REPORT/PGM/dis_dsover_s_t.sas OUT = REPORT/OUTPUT/dis_dsover_s_t_i.rtf (8 Mar. 2012-15:31)

TABLE 21 Analysis population SAR156597 10 20 40 80 150 300 Placebo mg mg mg mg mg mg All Safety population 12 6 6 6 6 6 6 48 Pharmacokinetic 0 6 6 5 6 6 6 35 population Pharmacodynamic 12 6 6 6 6 6 6 48 population PGM = PRODOPS/SAR156597/TDU11325/CSR/REPORT/PGM/dis_popover_a_t.sas OUT = REPORT/OUTPUT/dis_popover_a_t_i.rtf (8 Mar. 2012-15:31)

TABLE 22 Demographics and subject characteristics at baseline-safety population SAR156597 Placebo 10 mg 20 mg 40 mg 80 mg 150 mg 300 mg All (N = 12) (N = 6) (N = 6) (N = 6) (N = 6) (N = 6) (N = 6) (N = 48) Age (years) Number 12 6 6 6 6 6 6 48 Mean (SD) 31.7 (8.7) 29.5 (5.7) 29.0 (7.4) 30.5 (7.3) 33.0 (6.7) 30.0 (7.8) 29.3 (7.4) 30.6 (7.2) Median 30.0 29.5 25.0 33.0 34.5 29.0 27.5 30.0 Min:Max 22:44 22:39 24:42 19:37 21:41 21:41 21:40 19:44 Sex [n (%)] Number 12 6 6 6 6 6 6 48 Male 12 (100%) 6(100%) 6(100%) 6 (100%) 6 (100%) 6(100%) 6 (100%) 48 (100%) Race [n (%)] Number 12 6 6 6 6 6 6 48 Caucasian/White 9 (75.0%) 4 (66.7%) 5 (83.3%) 5 (83.3%) 4 (66.7%) 4 (66.7%) 4 (66.7%) 35 (72.9%) Black 1 (8.3%) 0 0 0 1 (16.7%) 0 2 (33.3%) 4 (8.3%) Asian/Oriental 1 (8.3%) 0 0 0 1 (16.7%) 0 0 2 (4.2%) Other 1 (8.3%) 2 (33.3%) 1 (16.7%) 1 (16.7%) 0 2 (33.3%) 0 7 (14.6%) Weight (kg) Number 12 6 6 6 6 6 6 48 Mean (SD) 76.23 78.55 71.67 79.85 79.30 77.92 80.32 77.51 (9.56) (10.50) (8.57) (8.95) (11.33) (8.39) (15.89) (10.22) Median 76.80 78.80 70.40 82.60 81.50 76.20 83.80 76.80 Min:Max 60.3:93.1 67.5:88.7 62.2:87.0 64.4:89.7 59.8:94.1 67.6:91.3 53.0:95.0 53.0:95.0 PGM = PRODOPS/SAR156597/TDU11325/CSR/REPORT/PGM/dem_dmsc_s_t.sas OUT = REPORT/OUTPUT/dem_dmsc_s_t_i.rtf (8 Mar. 2012-15:32)

TABLE 23 Overview of adverse event profile:treatment emergent adverse events-safety population SAR156597 Placebo 10 mg 20 mg 40 mg 80 mg 150 mg 300 mg n(%) (N = 12) (N = 6) (N = 6) (N = 6) (N = 6) (N = 6) (N = 6) Subjects with any TEAE 11 3 4 4 4 5 3 (91.7%) (50.0%) (66.7%) (66.7%) (66.7%) (83.3%) (50.0%) Subjects with any severe 1 0 0 0 0 0 0 TEAE (8.3%) Subjects with any treatment 0 0 0 0 0 1 0 emergent SAE (16.7%) Subjects with any TEAE na na na na na na na leading to permanent treatment discontinuation EAE: Treatment emergent adverse event, SAE: Serious adverse event na = not applicable N = Number of subjects treated within each group, n (%) = number and % of subjects with at least one TEAE in each category Note: An adverse event is considered as treatment emergent if it occurred from the time of the first investigational product (IP) administration up to the end of study visit (included). PGM = PRODOPS/SAR156597/TDU11325/CSR/REPORT/PGM/ae_aeover_s_t.sas OUT = REPORT/OUTPUT/ae_aeover_s_t_i.rtf (8 Mar. 2012-15:31)

TABLE 24 Number (%) of subjects with TEAE(s) by Primary SOC and PT-safety population SAR156597 Primary system organ class Placebo 10 mg 20 mg 40 mg 80 mg 150 mg 300 mg Preferred term [n (%)] (N = 12) (N = 6) (N = 6) (N = 6) (N = 6) (N = 6) (N = 6) Any class 11 (91.7%) 3 (50.0%) 4 (66.7%) 4 (66.7%) 4 (66.7%) 5 (83.3%) 3 (50.0%) Infections and infestations 2 (16.7%) 2 (33.3%) 1 (16.7%) 1 (16.7%) 2 (33.3%) 1 (16.7%) 2 (33.3%) Infectious mononucleosis 0 0 0 0 0 0 1 (16.7%) Upper respiratory tract infection 2 (16.7%) 1 (16.7%) 0 1 (16.7%) 0 0 1 (16.7%) Bronchitis 0 0 0 0 0 1 (16.7%) 0 Conjunctivitis infective 0 0 0 0 1 (16.7%) 0 0 Gastroenteritis 0 1 (16.7%) 0 0 0 0 0 Impetigo 0 0 0 0 1 (16.7%) 0 0 Pharyngitis 0 1 (16.7%) 1 (16.7%) 0 0 0 0 Blood and lymphatic system 0 0 0 1 (16.7%) 0 0 1 (16.7%) disorders Neutropenia 0 0 0 0 0 0 1 (16.7%) Eosinophilia 0 0 0 1 (16.7%) 0 0 0 Metabolism and nutrition disorders 0 0 1 (16.7%) 0 0 0 0 Decreased appetite 0 0 1 (16.7%) 0 0 0 0 Nervous system disorders 4 (33.3%) 1 (16.7%) 0 2 (33.3%) 2 (33.3%) 2 (33.3%) 0 Amnesia 0 0 0 1 (16.7%) 0 0 0 Dizziness 1 (8.3%) 0 0 0 0 1 (16.7%) 0 Headache 3 (25.0%) 1 (16.7%) 0 2 (33.3%) 2 (33.3%) 2 (33.3%) 0 Muscle contractions involuntary 1 (8.3%) 0 0 0 0 0 0 Presyncope 1 (8.3%) 0 0 0 0 0 0 Sinus headache 0 0 0 1 (16.7%) 0 0 0 Cardiac disorders 0 1 (16.7%) 0 0 0 0 0 Palpitations 0 1 (16.7%) 0 0 0 0 0 Vascular disorders 0 1 (16.7%) 0 0 0 1 (16.7%) 0 Deep vein thrombosis 0 0 0 0 0 1 (16.7%) 0 Orthostatic hypotension 0 1 (16.7%) 0 0 0 0 0 Respiratory, thoracic and 2 (16.7%) 2 (33.3%) 1 (16.7%) 1 (16.7%) 0 2 (33.3%) 1 (16.7%) mediastinal disorders Cough 1 (8.3%) 0 0 0 0 0 1 (16.7%) Dyspnoea 1 (8.3%) 1 (16.7%) 0 0 0 0 0 Nasal congestion 0 1 (16.7%) 1 (16.7%) 1 (16.7%) 0 1 (16.7%) 0 Oropharyngeal pain 0 0 0 0 0 1 (16.7%) 0 Pneumothorax 0 0 0 0 0 1 (16.7%) 0 Gastrointestinal disorders 3 (25.0%) 0 0 0 2 (33.3%) 3 (50.0%) 1 (16.7%) Constipation 0 0 0 0 0 1 (16.7%) 1 (16.7%) Diarrhoea 1 (8.3%) 0 0 0 0 0 1 (16.7%) Abdominal pain 0 0 0 0 1 (16.7%) 0 0 Dyspepsia 1 (8.3%) 0 0 0 0 0 0 Inguinal hernia 1 (8.3%) 0 0 0 0 0 0 Nausea 1 (8.3%) 0 0 0 0 2 (33.3%) 0 Toothache 0 0 0 0 1 (16.7%) 0 0 Vomiting 0 0 0 0 0 1 (16.7%) 0 Skin and subcutaneous tissue 2 (16.7%) 0 0 1 (16.7%) 0 1 (16.7%) 0 disorders Dermatitis contact 2 (16.7%) 0 0 1 (16.7%) 0 1 (16.7%) 0 Papule 0 0 0 0 0 1 (16.7%) 0 Musculoskeletal and connective 2 (16.7%) 0 2 (33.3%) 1 (16.7%) 1 (16.7%) 0 1 (16.7%) tissue disorders Back pain 0 0 1 (16.7%) 1 (16.7%) 0 0 1 (16.7%) Musculoskeletal stiffness 0 0 0 0 0 0 1 (16.7%) Myalgia 1 (8.3%) 0 1 (16.7%) 0 1 (16.7%) 0 0 Myositis 1 (8.3%) 0 0 0 0 0 0 Neck pain 0 0 1 (16.7%) 1 (16.7%) 0 0 0 Renal and urinary disorders 0 0 1 (16.7%) 0 0 0 0 Haematuria 0 0 1 (16.7%) 0 0 0 0 General disorders and 2 1 1 (16.7%) 0 1 (16.7%) 0 0 administration site conditions (16.7%) (16.7%) Fatigue 1 (8.3%) 0 0 0 1 (16.7%) 0 0 Influenza like illness 1 (8.3%) 0 0 0 0 0 0 Injection site erythema 1 (8.3%) 0 0 0 0 0 0 Pyrexia 0 1 (16.7%) 1 (16.7%) 0 0 0 0 Investigations 2 (16.7%) 0 1 (16.7%) 0 0 0 2 (33.3%) Aspartate aminotransferase 0 0 0 0 0 0 1 (16.7%) increased Blood creatine phosphokinase 1 (8.3%) 0 1 (16.7%) 0 0 0 1 (16.7%) increased C-reactive protein increased 0 0 0 0 0 0 1 (16.7%) Liver function test abnormal 0 0 0 0 0 0 1 (16.7%) Activated partial thromboplastin 1 (8.3%) 0 0 0 0 0 0 time prolonged Transaminases increased 1 (8.3%) 0 0 0 0 0 0 Injury, poisoning and procedural 3 (25.0%) 0 1 (16.7%) 0 1 (16.7%) 3 (50.0%) 0 complications Arthropod bite 1 (8.3%) 0 0 0 0 1 (16.7%) 0 Contusion 0 0 1 (16.7%) 0 1 (16.7%) 0 0 Excoriation 1 (8.3%) 0 1 (16.7%) 0 0 2 (33.3%) 0 Laceration 1 (8.3%) 0 0 0 0 0 0 Multiple fractures 0 0 0 0 0 1 (16.7%) 0 Muscle strain 0 0 0 0 0 1 (16.7%) 0 Road traffic accident 0 0 1 (16.7%) 0 0 1 (16.7%) 0 TEAE: Treatment emergent adverse event, SOC: Sytem organ class, PT: Preferred term MedDRA 14.1 N = Number of subjects treated within each group, n (%) = number and % of subjects with at least one TEAE in each category Note: Table sorted by SOC internationally agreed order and decreasing frequency of PT in SAR156597 300 mg group Note: An adverse event is considered as treatment emergent if it occurred from the time of the first investigational product (IP) administration up to the end of study visit (included). PGM = PRODOPS/SAR156597/TDU11325/CSR/REPORT/PGM/ae_iaeteae_s_t.sas OUT = REPORT/OUTPUT/ae_iaeteae_s_t_i.rtf (8 Mar. 2012-15:32)

TABLE 25 Hematology-Number of subjects with abnormalities (PCSA) during the TEAE period according to baseline status-safety population SAR156597 Placebo 10 mg 20 mg 40 mg 80 mg 150 mg 300 mg (N = 12) (N = 6) (N = 6) (N = 6) (N = 6) (N = 6) (N = 6) Laboratory parameter Nor. Abn. Nor. Abn. Nor. Abn. Nor. Abn. Nor. Abn. Nor. Abn. Nor. Abn. PCSA criteria n/N1 bas. bas. bas. bas. bas. bas. bas. bas. bas. bas. bas. bas. bas. bas. Hemoglobin 1/12 na 1/6 na 0/6 na 0/6 na 0/6 na 1/6 na 0/6 na Decr. from B ≥20 g/L Platelet count (thrombocyte 0/12 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 count) <100 Giga/L White blood cell count 0/12 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 (leukocyte count) <3.0 Giga/L (Non-Black); <2.0 Giga/L (Black) Neutrophils 0/12 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 1/6 0/0 <1.5 Giga/L (Non-Black); <1.0 Giga/L (Black) Eosinophils 0/12 0/0 0/6 0/0 0/6 0/0 0/5 1/1 1/6 0/0 0/6 0/0 0/6 0/0 >0.5 Giga/L or > ULN (if ULN ≥0.5 Giga/L) PCSA: Potentially Clinically Significant Abnormalities (Version of 14 Sep. 2009) LLN/ULN = Lower/Upper Limit of Normal range, B = Baseline, Nor. bas. = Normal baseline, Abn. bas. = Abnormal baseline (LLN/ULN or PCSA), Miss. bas. = Missing baseline, na = not applicable n/N1 = number of subjects who met the criterion at least once/number of subjects within each group who had that parameter assessed For hemoglobin, baseline values <LLN or >ULN (or LLN/ULN missing) are counted in one unique group (ie as normal), for eosinophils, values <LLN (or LLN missing) are counted as normal. Note: A PCSA is considered to be on-treatment if it occurred from the time of the first investigational product (IP) administration up to the end of study visit (included). PGM = PRODOPS/SAR156597/TDU11325/CSR/REPORT/PGM/lab_lbpcsa_s_t.sas OUT = REPORT/OUTPUT/lab_lbpcsa_s_t_hem_i.rtf (8 Mar. 2012-15:33)

TABLE 26 Biochemistry-Number of subjects with abnormalities (PCSA) during theTEAE period according to baseline status-safety population SAR156597 Placebo 10 mg 20 mg 40 mg 80 mg 150 mg 300 mg (N = 12) (N = 6) (N = 6) (N = 6) (N = 6) (N = 6) (N = 6) Laboratory parameter Nor. Abn. Nor. Abn. Nor. Abn. Nor. Abn. Nor. Abn. Nor. Abn. Nor. Abn. PCSA criteria n/N1 bas. bas. bas. bas. bas. bas. bas. bas. bas. bas. bas. bas. bas. bas. Glucose ≤3.9 mmol/L and <LLN 1/12 0/0 116 0/0 0/5 111 0/6 0/0 1/6 0/0 0/6 0/0 1/5 0/1 ≥11.1 mmol/L 0/12 0/0 0/6 0/0 0/5 0/1 0/6 0/0 0/6 0/0 0/6 0/0 0/5 0/1 (unfasted); ≥7 mmol/L (fasted) Total cholesterol 0/8 0/4 0/6 0/0 0/6 0/0 0/4 0/2 0/5 0/1 0/6 0/0 0/4 0/2 ≥7.74 mmol/L Triglycerides 0/10 2/2 0/6 0/0 0/6 0/0 0/4 2/2 0/6 0/0 0/5 0/1 1/4 0/2 ≥4.6 mmol/L Creatine phospho kinase  >3 ULN 1/12 0/0 0/6 0/0 1/5 0/1 0/6 0/0 0/5 0/1 0/4 1/2 1/6 0/0 >10 ULN 1/12 0/0 0/6 0/0 0/5 0/1 0/6 0/0 0/5 0/1 0/4 0/2 1/6 0/0 Highly sensitive 1/12 0/0 1/ 0/0 0/6 0/0 0/6 0/0 0/6 0/0 1/6 0/0 1/6 0/0 c-reactive protein >2 ULN Sodium ≤129 mmol/L 0/12 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 ≥160 mmol/L 0/12 010 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 Potassium <3 mmol/L 0/12 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 ≥5.5 mmol/L 0/12 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 Creatinine ≥150 μmol/L (Adults) 0/12 0/0 0/6 0/0 0/6 0/0 016 0/0 0/6 0/0 0/6 0/0 0/6 0/0 ≥30% change from B 0/12 na 0/6 na 0/6 na 0/6 na 0/6 na 0/6 na 0/6 na ALT (SGPT-ALAT) 0/12 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 >3 ULN AST (SGOT-ASAT)  >3 ULN 1/12 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0  >5 ULN 1/12 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 >10 ULN 0/12 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 Alkaline phosphatase 0/12 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 0/6 0/0 >1.5 ULN Total bilirubin >1.5 ULN 0/12 0/0 0/6 0/0 0/6 0/0 1/5 1/1 0/6 0/0 0/6 0/0 0/6 0/0   >2 ULN 0/12 0/0 0/6 0/0 0/6 0/0 0/5 0/1 0/6 0/0 0/6 0/0 0/6 0/0 PCSA: Potentially Clinically Significant Abnormalities (Version of 14 Sep. 2009) LLN/ULN = Lower/Upper Limit of Normal range, B = Baseline, Nor. bas. = Normal baseline, Abn. bas. = Abnormal baseline (LLN/ULN or PCSA), Miss. bas. = Missing baseline, na = not applicable n/N1 = number of subjects who met the criterion at least once/number of subjects within each group who had that parameter assessed For % change creatinine, baseline values <LLN or >ULN (or LLN/ULN missing) are counted in one unique group (ie as normal), for CPK, ALT, AST, ALP and Total Bilirubin, values <LLN (or LLN missing) are counted as normal. Note: A PCSA is considered to be on-treatment if it occurred from the time of the first investigational product (IP) administration up to the end of study visit (included). PGM = PRODOPS/SAR156597/TDU11325/CSR/REPORT/PGM/lab_lbpcsa_s_t.sas OUT = REPORT/OUTPUT/lab_lbpcsa_s_t_bio_i.rtf (8 Mar. 2012-15:32)

TABLE 27 Listing of subjects with combined PCSAs for liver function - safety population No occurrence PCSA: Potentially Clinically Significant Abnormalities (Version of 14 Sep. 2009) ULN = Upper Limit of Normal range, r = rechecked values, B = Baseline value * = ALT > 3 ULN and Total bilirubin > 2 ULN during the study, with at least one of them being post dose # = Conjugated Bilirubin > 35% Total bilirubin and Total Bilirubin > 1.5 ULN on the same sample post dose +/++ = Abnormal value reaching a 1^(st)/2^(nd) upper PCSA limit Note: A PCSA is considered to be on-treatment if it occurred from the time of the first investigational product (IP) administration up to the end of study visit (included). PGM = PRODOPS/SAR156597/TDU11325/CSR/REPORT/PGM/lab_lbhep_s_l.sas OUT = REPORT/OUTPUT/lab_lbhep_s_l_i.rtf (08 Mar. 2012 - 15:33)

TABLE 28 Listing of hsCRP >10 mg/L for at least 72 hours Theor. Sample Lab. hsCRP Treatment group Subject Visit time Date Time code (mg/L) SAR156597 150 mg 840001039 D-1 2011 Jun. 7 09:57 840000276 0.8B D1 T8H 2011 Jun. 8 16:25 840000276 0.5 D2 T24H 2011 Jun. 9 08:25 840000276 0.5 D3 T48H 2011 Jun. 10 08:25 840000276 0.5 D4 T72H 2011 Jun. 11 08:38 840000276 0.7 D5 2011 Jun. 12 08:14 840000276 0.7 D8 2011 Jun. 15 08:53 840000276 0.8 D15 2011 Jun. 22 09:00 840000276 1.6 D29 2011 Jul. 6 10:10 840000276 18.8H+ r 2011 Jul. 14 11:44 840000276 13.9H+ D57 2011 Aug. 3 10:13 840000276 1.2 EOS 2011 Aug. 31 10:25 840000276 0.9 r 2011 Oct. 13 10:36 840000276 2.2 FUP 2011 Dec. 1 10:56 840000276 1.3 r 2012 Feb. 6 10:59 840000276 0.8 SAR156597 300 mg 840001046 D-1 2011 Nov. 15 08:05 840000276 0.4B D1 T8H 2011 Nov. 16 17:35 840000276 0.4 D2 T24H 2011 Nov. 17 09:35 840000276 0.3 D3 T48H 2011 Nov. 18 09:35 840000276 0.2 D4 T72H 2011 Nov. 19 09:35 840000276 0.6 D5 2011 Nov. 20 09:35 840000276 0.6 D8 2011 Nov. 23 08:14 840000276 0.3 D15 2011 Nov. 30 08:21 840000276 0.3 D29 2011 Dec. 14 08:21 840000276 0.7 D57 2012 Jan. 11 08:25 840000276 0.3 EOS 2012 Feb. 8 08:32 840000276 37.0H+ r 2012 Feb. 10 07:55 840000276 91.5H+ r 2012 Feb. 13 08:17 840000276 23.6H+ r 2012 Feb. 27 08:07 840000276 0.5 L or H: Abnormal value <LLN or >ULN, −/−− or +/++: Abnormal value reaching a 1st/2nd lower or a 1st/2nd upper PCSA limit PCSA: Potentially Clinically Significant Abnormalities (Version of 14 Sep. 2009) LLN/ULN = Lower/Upper Limit of Normal range, r = rechecked values, B = Baseline value Baseline is Day-1 PGM = PRODOPS/SAR156597/TDU11325/CSR/REPORT/PGM/lab_hscrp_s_l.sas OUT = REPORT/OUTPUT/lab_hscrp_s_1_1_i.rtf (8 Mar. 2012-15:34)

TABLE 29 Listing of subjects with hsCRP >20 mg/L for 2 consecutive blood collections >=24 hours apart Sample Lab. hsCRP Treatment group Subject Visit Theor. Date Time code (mg/L) SAR156597 300 mg 840001046 D-1 time 2011 Nov. 15 08:05 840000276 0.4B D1 T8H 2011 Nov. 16 17:35 840000276 0.4 D2 T24H 2011 Nov. 17 09:35 840000276 0.3 D3 T48H 2011 Nov. 18 09:35 840000276 0.2 D4 T72H 2011 Nov. 19 09:35 840000276 0.6 D5 2011 Nov. 20 09:35 840000276 0.6 D8 2011 Nov. 23 08:14 840000276 0.3 D15 2011 Nov. 30 08:21 840000276 0.3 D29 2011 Dec. 14 08:21 840000276 0.7 D57 2012 Jan. 11 08:25 840000276 0.3 EOS 2012 Feb. 8 08:32 840000276 37.0H+ r 2012 Feb. 10 07:55 840000276 91.5H+ r 2012 Feb. 13 08:17 840000276 23.6H+ r 2012 Feb. 27 08:07 840000276 0.5 PGM = PRODOPS/SAR156597/TDU11325/CSR/REPORT/PGM/lab_hscrp_s_l.sas OUT = REPORT/OUTPUT/lab_hscrp_s_1_2_i.rtf (8 Mar. 2012-15:34)

TABLE 30 Listing of subjects with cTnI > 2ULN No occurrence L or H: Abnormal value < LLN or > ULN, −/−− or +/++: Abnormal value reaching a 1st/2nd lower or a 1st/2nd upper PCSA limit cTnI = Cardiac Troponin I PGM = PRODOPS/SAR156597/TDU11325/CSR/REPORT/PGM/lab_lbctnl_s_l.sas OUT = REPORT/OUTPUT/lab_lbctnl_s_l_i.rtf (08 Mar. 2012 - 15:34)

TABLE 31 Vital signs-Number of subjects with abnormalities (PCSA) during the TEAE period-safety population SAR156597 Vital signs parameter Placebo 10 mg 20 mg 40 mg 80 mg 150 mg 300 mg PCSA criteria n/N1 (N = 12) (N = 6) (N = 6) (N = 6) (N = 6) (N = 6) (N = 6) Systolic blood pressure ≤95 mmHg and decr. from B ≥20 mmHg 0/12 0/6 0/6 0/6 0/6 0/6 0/6 ≥140 mmHg and incr. from B ≥20 mmHg 0/12 0/6 0/6 0/6 0/6 0/6 1/6 Diastolic blood pressure ≤45 mmHg and decr. from B ≥10 mmHg 0/12 1/6 0/6 0/6 0/6 0/6 0/6 ≥90 mmHg and incr. from B ≥10 mmHg 0/12 0/6 0/6 0/6 0/6 0/6 0/6 Orthostatic systolic blood pressure 0/12 1/6 1/6 1/6 0/6 1/6 1/6 ≤−20 mmHg Orthostatic diastolic blood pressure 0/12 1/6 1/6 1/6 1/6 1/6 1/6 ≤-10 mmHg Heart rate ≤40 bpm and decr. from B ≥20 bpm 0/12 0/6 0/6 0/6 0/6 0/6 0/6 ≥100 bpm and incr. from B ≥20 bpm 0/12 0/6 0/6 0/6 0/6 0/6 016 Weight ≥5% decr. from B 2/12 0/6 0/6 0/6 016 0/6 0/6 ≥5% incr. from B 0/12 0/6 0/6 0/6 2/6 0/6 0/6 PCSA: Potentially Clinically Significant Abnormalities (Version of 14 Sep. 2009) decr./incr. = decrease/increase, B = Baseline n/N1 = Number of subjects who met the criterion at least once/number of subjects within each group who had that parameter assessed Note: A PCSA is considered to be on-treatment if it occurred from the time of the first investigational product (IP) administration up to the end of study visit (included). Orthostatic = standing after 3 minutes-supine after 10 minutes PGM = PRODOPS/SAR156597/TDU11325/CSR/REPORT/PGM/osa_vspcsa_s_t.sas OUT = REPORT/OUTPUT/osa_vspcsa_s_t_i.rtf (8 Mar. 2012-15:34)

TABLE 32 ECG-Number of subjects with abnormalities (PCSA) during the TEAE period-safety population ECG parameter (automatic SAR156597 reading) Placebo 10 mg 20 mg 40 mg 80 mg 150 mg 300 mg PCSA criteria n/N1 (N = 12) (N = 6) (N = 6) (N = 6) (N = 6) (N = 6) (N = 6) Heart rate ≤40 bpm and decr. from B ≥20 0/12 0/6 0/6 0/6 0/6 0/6 0/6 bpm ≥100 bpm and incr. from B ≥20 0/12 0/6 0/6 0/6 0/6 0/6 0/6 bpm PR interval 1/12 1/6 0/6 0/6 0/6 0/6 0/6 ≥220 ms QRS interval 1/12 0/6 2/6 0/6 0/6 0/6 0/6 ≥120 ms QTc interval Borderline: 431-450 ms (Male); 3/12 0/6 0/6 2/6 1/6 3/6 0/6 451-470 ms (Female) Prolonged: >450 ms (Male); 0/12 0/6 0/6 0/6 0/6 0/6 0/6 >470 ms (Female) ≥500 ms 0/12 0/6 0/6 0/6 0/6 0/6 0/6 QTc interval-change from baseline Borderline: Incr. from B 30-60 ms 2/12 216 1/6 1/6 1/6 0/6 0/6 Prolonged: Incr. from B >60 ms 0/12 0/6 0/6 0/6 0/6 0/6 0/6 PCSA: Potentially Clinically Significant Abnormalities (Version of 14 Sep. 2009) decr./incr. = decrease/increase, B = Baseline n/N1 = number of subjects who met the criterion at least once/number of subjects within each group who had that parameter assessed Note: A PCSA is considered to be on-treatment if it occurred from the time of the first investigational product (IP) administration up to the end of study visit (included). PGM = PRODOPS/SAR156597/TDU11325/CSR/REPORT/PGM/osa_egpcsa_s_t.sas OUT = REPORT/OUTPUT/osa_egpcsa_s_t_i.rtf (8 Mar. 2012-15:33)

TABLE 33 Listing of subjects with prolonged QTc and/or delta QTc > 60 ms (automatic reading) - safety population No occurrence PCSA: Potentially Clinically Significant Abnormalities (Version of 14 Sep. 2009) B = Baseline, Delta = change from baseline (B), r = rechecked values − or +/++: Abnormal value reaching the lower or a 1^(st)/2^(nd) upper PCSA limit Note: Baseline is defined as the mean of triplicates at D1 predose. Note: A PCSA is considered to be on-treatment if it occurred from the time of the first investigational product (IP) administration up to the end of study visit(included). PGM = PRODOPS/SAR156597/TDU11325/CSR/REPORT/PGM/osa_egauprol_s_l.sas OUT = REPORT/OUTPUT/osa_egauprol_s_l_i.rtf (08 Mar. 2012 - 15:33)

TABLE 34 Total IgE-Descriptive statistics Raw data % Change from baseline N Mean SD SEM Median Min Max N Mean SD SEM Median Min Max Placebo Baseline 12 80.70 109.71 31.672 35.75 1.0 397.0 D2 T24H 12 85.87 121.41 35.049 36.90 1.0 435.0 12 2.26 11.32 3.267 3.27 −25.6 19.7 D15 12 82.64 112.33 32.427 35.40 1.0 393.0 12 −1.40 11.83 3.416 −0.84 −19.2 30.3 D29 12 76.58 109.84 31.709 34.95 1.0 402.0 12 −6.06 10.09 2.912 −2.98 −23.2 6.0 EOS 12 76.26 103.95 30.007 32.70 1.0 386.0 12 7.74 44.99 12.988 −1.39 −31.0 142.4 SAR156597 10 mg Baseline 6 26.18 11.65 4.754 24.70 14.3 40.1 D2 T24H 6 26.67 11.66 4.760 25.50 14.4 39.8 6 2.12 3.13 1.280 0.67 −0.7 7.8 D15 6 23.27 9.44 3.854 20.15 14.8 38.5 6 −7.25 17.53 7.157 −3.27 −41.8 7.7 D29 6 22.43 9.01 3.678 19.90 14.5 38.1 6 −10.02 18.40 7.511 −5.72 −45.0 5.6 EOS 6 23.55 9.76 3.983 22.05 14.1 39.2 6 −7.51 13.14 5.365 −4.31 −33.1 4.4 SAR156597 20 mg Baseline 6 198.60 434.00 177.179 27.30 1.0 1084.0 D2 T24H 6 192.57 420.30 171.585 27.60 1.0 1050.0 6 −1.85 5.59 2.284 −2.00 −8.9 7.4 D15 6 163.95 352.56 143.931 23.85 1.0 883.0 6 −8.53 8.76 3.578 −8.13 −18.5 2.0 D29 6 161.83 346.71 141.545 25.35 1.0 869.0 6 −6.62 7.02 2.865 −4.80 −19.8 0.0 EOS 6 179.87 393.19 160.519 23.50 1.0 982.0 6 −8.19 7.64 3.118 −6.24 −20.0 0.0 SAR156597 40 mg Baseline 6 134.77 191.25 78.076 67.50 19.2 516.0 D2 T24H 6 139.22 202.79 82.789 64.70 19.8 545.0 6 1.61 4.05 1.653 2.68 −6.2 5.6 D15 5 125.94 178.28 79.730 31.00 20.5 437.0 5 −1.33 8.59 3.841 1.75 −15.3 6.8 D29 5 125.32 186.47 83.390 31.30 19.2 454.0 5 −5.70 6.91 3.091 −2.19 −14.3 0.0 EOS 6 129.82 172.61 70.467 73.85 25.0 477.0 6 28.14 85.72 34.994 −4.40 −14.4 202.6 SAR156597 80 mg Baseline 6 56.05 44.60 18.207 50.85 7.2 118.0 D2 T24H 6 56.03 46.43 18.954 50.10 3.8 119.0 6 −7.61 19.58 7.995 0.63 −47.2 3.8 D15 6 53.33 40.62 16.585 56.65 3.9 103.0 6 −9.13 21.02 8.582 −7.95 −45.8 17.7 D29 6 56.83 44.87 18.320 60.00 3.0 114.0 6 −6.92 28.41 11.598 −4.14 −58.3 29.2 EOS 6 56.60 43.95 17.942 59.95 2.8 114.0 6 −2.35 36.51 14.904 −7.33 −61.1 37.4 SAR156597 150 mg Baseline 6 38.67 28.37 11.582 32.45 13.0 93.8 D2 T24H 6 38.67 27.72 11.315 31.40 11.7 91.0 6 −0.47 9.00 3.674 −2.91 −10.0 16.6 D15 6 36.07 26.61 10.862 30.95 10.6 87.1 6 −7.95 6.29 2.569 −8.09 −18.5 0.0 D29 6 47.43 51.09 20.857 31.15 12.1 150.0 6 7.12 26.56 10.844 −1.30 −11.4 59.9 EOS 6 39.95 29.18 11.912 30.65 13.0 96.4 6 3.86 12.13 4.952 4.23 −16.3 20.0 SAR156597 300 mg Baseline 6 51.15 62.93 25.689 24.45 20.0 179.0 D2 T24H 6 48.52 61.22 24.992 21.60 20.3 173.0 6 −6.57 6.20 2.532 −6.57 −16.9 2.0 D15 6 37.43 42.19 17.223 21.75 14.4 123.0 6 −21.12 18.99 7.753 −28.65 −31.3 17.4 D29 6 36.47 39.32 16.051 20.85 15.1 116.0 6 −21.67 16.26 6.637 −26.71 −35.2 10.1 EOS 6 50.30 56.09 22.900 22.95 13.7 158.0 6 0.82 38.97 15.909 −10.03 −31.5 78.0 N corresponds to the count of subjects with available data Baseline is the Day-1 value Values below LOQ (Limit Of Quantification) were replaced by LOQ/2 PGM = PRODOPS/SAR156597/TDU11325/CSR/REPORT/PGM/pd_lbsum_s_t.sas OUT = REPORT/OUTPUT/pd_lbsum_s_t_1_i.rtf (8 Mar. 2012-15:35

TABLE 35 Pharmacokinetic parameters for plasma concentration of SAR156597 after a single subcutaneous dose given to young healthy male subjects (TDU11325) Mean ± SD (Geometric Mean) [CV %] Plasma SAR156597 10 mg 20 mg 40 mg 80 mg 150 mg 300 mg N 6 6 5 6 6 6 C_(max) 0.971 ± 1.69 ± 3.32 ± 7.01 ± 1.97 9.44 ± 1.78 24.1 ± 4.60 0.254 0.222 0.797 (μg/ml) (0.943) (1.67) (3.25) (6.81) (9.29) (23.8) [19.0] [26.1] [13.1] [24.0] [28.1] [18.9] t_(max) ^(a) 108 96 119 144 168 96 (hr) (48-168) (73-170) (48-240) (72-240) (120-240) (71-168) t_(1/2z) 369 ± 106 350 ± 54.4 320 ± 34.8 372 ± 104 464 ± 72.0 315 ± 71.4 (hr) (355) [28.6] (345) (319) (358) (460) [15.5] (308) [22.7] [15.6] [10.9] [27.9] AUC_(last) 607 ± 174 1000 ± 295 1920 ± 3670 ± 5570 ± 761 11300 ± 377 1100 2900 (μg · hr/ml) (587) [28.7] (967) (1890) (3530) (5530) (10900) [29.4] [19.6] [30.0] [13.7] [25.7] AUC 644 ± 185 1050 ± 1960 ± 3780 ± 5820 ± 844 11500 ± 297 378 1140 3000 (μg · hr/ml) (622) [28.7] (1010) (1930) (3630) (5770) (11100) [28.3] [19.3] [30.2] [14.5] [26.1] V_(ss)/F 9230 ± 11400 ± 10700 ± 11400 ± 16500 ± 13100 ± 2400 2370 1740 2870 2270 2300 (ml) (8990) (11100) (10600) (11100) (16300) (12900) [26.0] [20.9] [16.2] [25.1] [13.8] [17.6] CL/F 16.6 ± 4.55 20.4 ± 21.0 ± 23.0 ± 7.62 26.2 ± 3.66 28.2 ± 10.2 5.86 4.05 (ml/hr) (16.1) (19.7) (20.7) (22.0) (26.0) (27.0) [36.2] [27.4] [28.7] [19.2] [33.1] [14.0] t_(last) ^(a) 1679 1680 1704 2016 2020 2016 (hr) (1008-2016) (1345-2016) (1633-2016) (1368-2018) (2014-2111) (1344-2039) ^(a)Median (Min-Max) NA = Not Applicable Source = PKS Study: TDU11325 DBLII E02; Scenario: P-D-EV-SD-E02, Version 1 Date/Time = Mar. 6, 2012 11:25:36 AM 

1. A maximal safe therapeutic dose of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 to a human subject having an area under the plasma concentration versus time curve calculated using the trapezoidal method from time zero to real time (AUC_(last)) from about 433 ug·h/ml to about 14200 ug·h/ml.
 2. A maximal safe therapeutic dose of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 to a human subject having an area under the plasma concentration versus time curve extrapolated to infinity (AUC) from about 459 ug·h/ml to about 670014500 ug·h/ml.
 3. A maximal safe therapeutic dose of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 to a human subject having a maximum plasma concentration observed (C_(max)) from about 0.717 ug/ml to about 28.7 ug/ml.
 4. A maximal safe therapeutic dose of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 to a human subject having a first time to reach a maximum plasma concentration (t_(max)) from about 96 hr to about 168 hr.
 5. A method monitoring whether a therapeutic dose of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 administered to a human subject is safe, said method comprising: (a) administering said therapeutic dose of said dual V region antibody-like protein or a fragment of a dual V region antibody-like region to said human subject; (b) measuring one or more events selected from the group consisting of intensive treatment in an emergency room or at home for allergic bronchospasm, blood dyscrasias, convulsions, alanine aminotransferase (ALT) >3×upper limit of normal range (ULN) associated with total bilirubin >2×ULN, asymptomatic ALT increase >10×ULN, development of drug dependency or drug abuse, ALT increase ≥2×ULN, hsCRP >10 mg/L for ≥72 hours, cardiac troponin I (cTnI) >2×ULN, a ventricular depolarization and repolariztion time (QT) on an electrocardiogram (ECG) machine wherein the QT is automatically corrected by the ECG machine (QTc) that is QTc ≥500 ms and severe skin reactions local to the site of IP injection; and (c) determining one or more said events as measured in (b) has occurred wherein said therapeutic dose is identified as not safe and the therapeutic dose is discontinued or lowered.
 6. A method of selecting a safe therapeutic dose or of monitoring the safe use of a therapeutic dose of dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 to a human subject, said method comprising: (a) administering a dose of said dual V region antibody-like protein or a fragment of a dual V region antibody-like region to said human subject; (b) measuring a level of C-reactive protein (CRP) in a blood sample from said human subject; and (c) determining said level of C-reactive protein (CRP) is less than 20 mg/L as measured in (b) wherein said dose is selected as said safe therapeutic dose to be administered to said human subject.
 7. A method of selecting a safe therapeutic dose or of monitoring the safe use of a therapeutic dose of a dual V region antibody-like protein or a fragment of a dual V region antibody-like region that specifically binds to IL-4 and IL-13 to a human subject, said method comprising: (a) administering a dose of said dual V region antibody-like protein or a fragment of a dual V region antibody-like region to said human subject; (b) measuring a ventricular depolarization and repolariztion time (QT) on an electrocardiogram (ECG) machine wherein the QT is automatically corrected by the ECG machine (QTc) of said human subject; and (c) determining said QTC is less than 500 ms as measured in (b) wherein said dose is selected as said safe therapeutic dose to be administered to said human subject.
 8. The method of any one of the preceding claims, wherein the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a variable light chain comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO: 3 and a variable heavy chain comprising amino acid sequences SEQ ID NO:2 and SEQ ID NO:
 4. 9. The method of any one of the preceding claims, wherein the dual V region antibody-like protein or the fragment of a dual V region antibody-like region comprises a peptide linker such that SEQ ID NO:1 and SEQ ID NO:3 are linked together with the peptide linker and SEQ ID NO:2 and SEQ ID NO:4 are linked together with the peptide linker.
 10. The method of any one of the preceding claims, wherein the peptide linker consists of SEQ ID NO:
 6. 11. The method of any one of the preceding claims, wherein the therapeutic dose is equal to or less than about 300 mg.
 12. The method of any one of the preceding claims, wherein the therapeutic dose is selected from the group consisting of 10 mg, 20 mg, 40 mg, 80 mg, 150 mg and 300 mg. 